Method for administering camptothecins VIA injection of pharmaceutical composition comprising coated particles of a camptothecin

ABSTRACT

A method for administering a camptothecin to a patient comprising: injecting into a patient a pharmaceutical composition comprising an aqueous suspension of solid particles suitable for intravenous delivery, the solid particles comprising a camptothecin, and a 0.3 nm to 3.0 μm thick layer of a membrane-forming amphipathic lipid wherein the solid particles have mean diameters between about 0.05 μm and 10 μm. The method is particularly well suited for delivering camptothecins, particularly 9-nitro-camptothecin intravenously.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to compositions and methods for deliveringcamptothecin, in particular, 9-nitro-camptothecin, to patients byinjection and treating diseases associated with abnormal cellproliferation such as cancer.

[0003] 2. Description of Related Art

[0004] Camptothecin was isolated from the plant, Camptotheca acuminata,in the 1960's (Wall, M. et al. (1966) J. Am. Chem. Soc. 88: 3888-3890).Camptothecin has a pentacyclic ring system with only one asymmetriccenter in ring E with a 20(S)-configuration. The pentacyclic ring systemincludes a pyrrole quinoline moiety (rings A, B and C), a conjugatedpyridone (ring D), and a six-membered lactone (ring E) with an(α-hydroxyl group (i.e., an α-hydroxy lactone).

[0005] Camptothecin itself is highly lipophilic and poorlywater-soluble. Sodium camptothecin that is solubilized by sodiumhydroxide in water was used in clinical trials in the early 70's andfound to have antitumor activity. However, this formulation ofcamptothecin administered via i.v. caused unpredictable side effectssuch as myelosuppression and hemorrhagic cystitis. Clinical trials withsodium camptothecin were eventually discontinued because of thesetoxicities and the lack of consistent antitumor activity.

[0006] Continued evaluation of this agent showed that the sodiumcarboxylate salt is only 10% as potent as the native camptothecin withthe closed α-hydroxy lactone ring intact (Wall et al. in (1969)“International Symposium on Biochemistry and Physiology of theAlkaloids, Mothes et al. eds. Academic Verlag, Berlin, 77; Giovanella etal. (1991) Cancer Res. 51:3052). Studies also showed that camptothecinand its derivatives undergo an alkaline hydrolysis of the E-ringα-hydroxy lactone, resulting in a carboxylate form of camptothecin. AtpH levels below 7.0, the α-hydroxy lactone E-ring form of camptothecinpredominates. However, intact lactone ring E and α-hydroxyl group havebeen shown to be essential for antitumor activity of camptothecin andits derivatives.

[0007] Camptothecin and its derivatives have been shown to inhibit DNAtopoisomerase I by stabilizing the covalent complex (“cleavablecomplex”) of enzyme and strand-cleaved DNA. Inhibition of topoisomeraseI by camptothecin induces protein-associated DNA single-strandbreakswhich occur during the S-phase of the cell cycle. Since the S-phase isrelatively short compared to other phases of the cell cycle, longerexposure to camptothecin should result in increased cytotoxicity oftumor cells. Studies indicate that only the closed α-hydroxy lactoneform of the drug helps stabilize the cleavable complex, leading toinhibition of the cell cycle and apoptosis.

[0008] To preserve the α-hydroxy lactone form of camptothecin,camptothecin and its water insoluble derivatives have been dissolved inN-methyl-2-pyrrolidinone in the presence of an acid (U.S. Pat. No.5,859,023). Upon dilution with an acceptable parenteral vehicle, astable solution of camptothecin was obtained. The concentrated solutionof camptothecin was also filled in gel capsules for oral administration.It is believed that such formulations increase the amount of lipophilicα-hydroxy lactone form of camptothecin that diffuse through the cellularand nuclear membranes in tumor cells.

[0009] T. G. Burke, A. E. Staubus, A. K. Mishra and H. Malak (“LiposomalStabilization of Camptothecin's Lactone Ring.” J. Am. Chem. Soc. 1992,114, 8318) and T. G. Burke, A. K. Mishra, M. C. Wani and M. E. Wall(“Lipid bilayer partitioning and stability of camptothecin drugs.”Biochemistry 1993 May 25;32 (20):5352-64) have demonstrated thatharboring of camptothecin drugs into phospholipid bilayer membranesstabilized the α-hydroxy lactone moiety of camptothecin drugs againsthydrolysis. In comparison to hydrolysis half-lives in PBS at 37° C. ofapproximately 15 to 30 min, lipid bilayer membrane-bound camptothecindrugs were found to be stable even for periods up to 72 hours. Theseauthors have determined an iodide ion induced quenching behavior ofcamptothecin's fluorescence indicative of intercalation of camptothecinmolecules between the phospholipid acyl chains of membrane bilayers, aprotected environment removed from the aqueous interface. The potentialfor stabilization of camptothecin's α-hydroxy lactone ring structure inthis environment led to the expectation that lipid bilayer intercalationmight conserve the biologically active form in vivo, thereby permittingthe active form to be delivered via liposomal bilayers into a biologicalhost (U.S. Pat. No.5,552,156).

[0010] Z Mi and T. G. Burke (“Differential interactions of camptothecinlactone and carboxylate forms with human blood components.” Biochemistry1994 August 30;33 (34):10325-36) exploited the intrinsic fluorescentemissions from the α-hydroxy lactone and carboxylate forms ofcamptothecin in order to elucidate their markedly different interactionswith the various components of human blood. It was found that in PBS atpH 7.4, human serum albumin (HSA) preferentially binds the carboxylateform of camptothecin with a 150-fold higher affinity than the α-hydroxylactone form. These interactions cause camptothecin's α-hydroxy lactonering to open more rapidly and completely in the presence of HSA than inthe protein's absence. In human plasma, at pH 7.4 and 37° C., they haveobserved camptothecin's α-hydroxy lactone ring to open rapidly and fullyto the carboxylate form (half-life=11 min; % α-hydroxy lactone atequilibrium, 0.2%). They concluded that camptothecin carboxylate'sfluorophore locates in a hydrophobic binding pocket in native HSA. Inwhole blood versus plasma, camptothecin's α-hydroxy lactone was found todisplay enhanced stability resulting in an increased half-life of 22 minand an equilibrium α-hydroxy lactone concentration of 5.3%. The enhancedstability of camptothecin in human blood was found to be due to drugassociations with the lipid bilayers of red blood cells. Althoughcamptothecin α-hydroxy lactone hydrolysis slows down and the equilibriumα-hydroxy lactone form concentration rises on intercalation withinphospholipid bilayers, the membrane-bound drug still remainsthermodynamically and kinetically too labile in the presence of albuminand the concentration of the active α-hydroxy lactone form in plasmaremains insufficient. Thus, the liposomal bilayers cannot be consideredas a pragmatic delivery system for this drug. It is speculated that aprincipal deactivation channel of membrane bilayer-bound camptothecin isfacilitated via high affinity of the carboxylate form of the drug withalbumin in-vivo compared to a relatively lower affinity of the α-hydroxylactone form of the drug with the membrane bilayers.

[0011] U.S. Pat. Nos. 5,552,156 and 5,736,156 describe liposomes andmicelles of surfactant molecules for intravenous delivery ofcamptothecins. In liposomes, the camptothecin can reside bound to andpartially in the membrane interlayer or dissociate into the internalenclosed aqueous layer in direct contact with water where thecamptothecin lactone is not stable to hydrolysis. In micelles ofsurfactant molecules, the camptothecin is either in the centralhydrocarbon portion of the micelle, bound to the micelle membrane orbound to the outside of the micelle. However, while camptothecins areless stable in micelles than in liposomes, especially in poly(ethyleneoxide)-containing micelles, the amount of camptothecin compound that canbind to the membrane layer in a liposome is limited to the dimensions ofthe membrane and to the requirement that the membrane remain intact toprevent rupture of the liposome. The ratio of lipid to camptothecin inliposomes is generally greater than 150, and the lactone of thecamptothecin slowly hydrolyzes because of the reported equilibriumbetween bound and free camptothecin.

SUMMARY OF THE INVENTION

[0012] The present invention provides novel injectable formulations ofcamptothecin, including analogs, derivatives, and pharmaceuticallyactive metabolites of 20(S)-camptothecin, referred to herein ascamptothecin compound. The present invention also provides methods ofmanufacturing these formulations, kits containing these formulations andmethods of using these formulations to treat patients having diseasesassociated with abnormal cell proliferation, such as cancer.

[0013] One class of injectable formulations comprise micron andsubmicron size particle suspensions of a camptothecin. These micron andsubmicron size particle suspensions of camptothecin substantially reducedeactivation of the camptothecin by hydrolysis in-vitro and by plasmacomponents in-vivo. The particle suspensions of the present inventiondisplay efficacy in the treatment of diseases associated with abnormalcell proliferation such as cancer when injected intravenously intomammals containing such diseases. The micron and submicron size particlesuspensions of α-hydroxy lactone-containing camptothecin drugs of thisinvention are preferably substantially stable to autoclaving, undergoingsterilization without suffering from hydrolytic deactivation of theα-hydroxy lactone or substantial change in particle size. The particlesuspensions can be autoclaved, cooled, and stored for long periodswithout undergoing agglomeration, flocculation, or aggregation.

[0014] In one embodiment, the injectable formulation comprises anaqueous suspension of solid particles suitable for intravenous delivery,the solid particles comprising a camptothecin, the solid particleshaving mean diameters between about 0.05 μm and 10 μm, the particlescoated with a 0.3 nm to 3.0 μm thick layer of a membrane-formingamphipathic lipid.

[0015] In another embodiment, the injectable pharmaceutical compositioncomprises an aqueous suspension of solid particles suitable forintravenous delivery, the solid particles comprising a camptothecin, anda 0.3 nm to 3.0 μm thick outer layer comprising a membrane-formingamphipathic lipid; wherein the solid particles have mean diametersbetween about 0.05 ,μm and 10 μm.

[0016] In yet another embodiment, an injectable pharmaceuticalcomposition is provided which comprises: a dispersion of micrometer tosubmicrometer size solid particles in an aqueous carrier solutioncomprising one or more pharmaceutically acceptable tonicity modifieragents, the solid particles comprising a camptothecin, a first coatingof not more than 10% w/w of a substantially water-insoluble,pharmaceutically acceptable lipophilic agent in which the camptothecindrug is insoluble or poorly soluble, and a second coating of at leastone membrane-forming surface stabilizing amphipathic lipid, wherein thedispersion does not aggregate, flocculate, or agglomerate, and theparticles do not grow in size above a volume weighted mean diameter of10 μm upon thermal sterilization.

[0017] In yet another embodiment, an injectable pharmaceuticalcomposition is provided which comprises: a dispersion of micrometer tosubmicrometer size solid particles in an aqueous carrier solutioncomprising one or more pharmaceutically acceptable tonicity modifieragents, the solid particles comprising: a camptothecin, and a coating ofat least one membrane-forming surface stabilizing amphipathic lipid,wherein the dispersion does not aggregate, flocculate, or agglomerate,and the particles do not grow in size above a volume weighted meandiameter of 10 μm upon thermal sterilization.

[0018] In yet another embodiment, a pharmaceutical composition suitablefor intravenous administration to a mammal is provided which comprises:an aqueous suspension of particles having mean diameters between about0.05 μm and 10 μm, the particles comprising a camptothecin having anintact lactone ring, a membrane-forming amphiphatic lipid coating anouter surface of the particle, and one or more tonicity modifyingagents, wherein the ratio of lipid to camptothecin in the particle isless than about 150:1 moles:mole, and wherein the mean diameters of theparticles increase less than 100% upon thermal sterilization.

[0019] Another class of injectable formulations comprise an aqueousdispersion of microdroplets suitable for intravenous delivery, themicrodroplets comprising a camptothecin. It is noted with regard to themicrodroplet formulations that the camptothecin may be present where itis dissolved in a water-insoluble, pharmacologically acceptable liquid.The camptothecin may also be present in a solid form in addition tobeing dissolved in the water-insoluble, pharmacologically acceptableliquid. For example, the camptothecin may be present in thewater-insoluble, pharmacologically acceptable liquid beyond thesolubility of the camptothecin in the liquid.

[0020] In one embodiment, the injectable pharmaceutical compositioncomprises: an aqueous suspension of microdroplets suitable forintravenous delivery, the microdroplets having a mean diameter between200 Angstroms and one micron, the microdroplets comprising asubstantially water-insoluble, pharmacologically acceptable liquid, acamptothecin dissolved in the water-insoluble, pharmacologicallyacceptable liquid, and an outer layer comprising a phospholipid.

[0021] In another embodiment, the injectable pharmaceutical compositioncomprises: a dispersion in an aqueous carrier solution comprising one ormore pharmaceutically acceptable tonicity modifier agents and liquiddroplets of micrometer to submicrometer, the droplets comprising asubstantially water-insoluble, pharmaceutically acceptable lipophilicliquid vehicle a camptothecin dissolved in the lipophilic liquidvehicle, and an outer layer surrounding the droplet comprising at leastone membrane-forming amphipathic lipid, wherein upon thermalsterilization the dispersion does not aggregate, flocculate,agglomerate, or coalesce, and the droplets do not grow in size above avolume weighted mean diameter of 10 μm.

[0022] In yet another embodiment, the injectable pharmaceuticalcomposition comprises: an aqueous carrier solution comprising one ormore pharmaceutically acceptable tonicity modifier agents; a dispersionof liquid droplets of a first size distribution, the liquid dropletscomprising a substantially water-insoluble, pharmaceutically acceptablelipophilic liquid vehicle, solid particles of a camptothecin of a secondsize distribution, and an outer layer surrounding the droplet comprisingat least one membrane-forming amphipathic lipid; wherein the first sizedistribution is in the range of submicrometer to micrometers, and thesecond size distribution is smaller than the first size distribution;and wherein upon thermal sterilization, the dispersion does notaggregate, flocculate, agglomerate, or coalesce, and the droplets do notgrow in size above a volume weighted mean diameter of 10 μm.

[0023] The camptothecins used in the formulations of the presentinvention may be an analog, a derivative or a pharmacologically activemetabolite of 20(S)-camptothecin. In one embodiment, the camptothecincompound in the formulation has poor water solubility, for examplehaving a water solubility of 10 μg/ml or less.

[0024] Examples of the camptothecins include, but are not limited to,20(S)-camptothecin, 9-nitro-20(S)-camptothecin,9-amino-20(S)-camptothecin,7-ethyl-10-(4-(1-piperdino)-1-piperdino)-carbonyloxy-camptothecin,7-ethyl-10-hydroxy-20(S)-camptothecin,10,11-methylenedioxy-20(S)-camptothecin, 9-chloro-20(S)-camptothecin,9-bromo-20(S)-camptothecin, 9-hydroxy-20(S)-camptothecin, and11-hydroxy-20(S)-camptothecin.

[0025] With regard to each of the formulations described herein, thecamptothecin compound preferably contains a α-hydroxy lactone ring andis most preferably 9-nitro-20(S)-camptothecin.

[0026] According to these various embodiments, the pharmaceuticalcomposition preferably has a pH less than 7, preferably a pH less than 6and in one embodiment, a pH between 5 and 6.

[0027] Also according to these various embodiments, the membrane-formingamphipathic lipid optionally comprises a phospholipid. The phospholipidmay optionally be selected from the group consisting of saturatedphospholipids, unsaturated phospholipids, synthetic phospholipids,natural phospholipids, and combinations thereof. The phospholipid mayoptionally be selected from the group consisting of natural andsynthetic lipids, hen egg-derived phospholipid, egg phospholipid,purified egg phospholipid, soy phospholipid, dimyristoyl lecithin,didodecanoyl lecithin, dioeoyl lecithin, dilinoeoyl lecithin,alpha-palmito-beta-oleoyl lecithin, alpha-palmitoyl-beta-linoleoyllecithin, alpha-oleoyl-beta-palmitoyl lecithin, diarachidonyl lecithin,alpha-palmito-beta-myristoyl lecithin, dimyristoyl phosphatidic acid,dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid,phosphatidyl serine, phosphatidyl inositol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidyl glycerol, dioctadecanoyl phosphatidylethanolamine, dioleoyl phosphatidyl ethanolamine, dihexadecylphosphatidyl ethanolamine, dilauryl phosphatidyl ethanolamine,dimyristoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidylethanolamine, Lipoid E80, Lipoid ES, Lipoid 90H, and Lipoid 100H. In oneparticular variation, the phospholipid comprises Lipoid E80.

[0028] Also according to these various embodiments, the outer layer mayfurther comprises cholesterol.

[0029] Also according to these various embodiments, the camptothecin maybe present in amounts of up to about 25% w/w, more preferably up toabout 5% w/w. The camptothecin may be present in amounts of from about0.05% w/w to about 5% w/w, and optionally from about 0.1% w/w to about1% w/w. In one particular variation, the camptothecin is present inamount of about 0.2% w/w.

[0030] Also according to these various embodiments, the membrane-formingamphipathic lipid may be present in amounts of from 0.2% w/w to about 5%w/w, more preferably in amounts of from 1% w/w to about 5% w/w. In oneparticular variation, the membrane-forming amphipathic lipid is presentin amounts of about 4% w/w.

[0031] According to each of the above embodiments, the mean diameters ofthe particles and droplets preferably increase less than 100% uponstorage at room temperature (23° C.) for at least 1 month, morepreferably at least 3 months, more at least 6 months, and mostpreferably at least 1 year.

[0032] Also according to each of the above embodiments, the lipophilicliquid vehicle is optionally present in amounts up to about 40% w/w,more preferably in amounts up to about 25% w/w, and most preferably inamounts up to about 20% w/w. The lipophilic liquid vehicle is alsopreferably present in amounts greater than 0.5% w/w.

[0033] The various compositions and kits of the present invention may beused in methods to treat patients suffering from abnormal cellproliferation such as cancer including hematological malignancy andtumors. In one embodiment, the method comprises: providing apharmaceutical composition according to the present invention; andadministering a therapeutically effective amount of the pharmaceuticalcomposition intravenously to a patient in need thereof. According to themethod, the camptothecin compound preferably contains a α-hydroxylactone ring and is preferably 9-nitro-20(S)-camptothecin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 describes IDD-P or IDD-D formulations which are used in thehuman cancer studies described herein.

[0035]FIG. 2 summarizes the volume weighted mean particle size and 99.9percentile data for the temperature stability study for IDD-P.

[0036]FIG. 3 summarizes the particle size stability data from stabilitystudies on IDD-D.

[0037]FIG. 4 summarizes the protocol design for the first 375 melanomastudy.

[0038]FIG. 5 summarizes the therapeutic and toxic responses for all micegroups in the first 375 melanoma study.

[0039]FIG. 6 illustrates a scatter plot showing the individual survivaltimes for the mice in all groups in the first 375 melanoma.

[0040]FIG. 7 shows Kaplan-Meier survival curves from the first melanomastudy for untreated mice and mice treated with 9-nitro-camptothecin inIDD-D or vehicle.

[0041]FIG. 8 shows the Kaplan-Meier survival curves from the firstmelanoma study for untreated mice and mice treated with oral9-nitro-camptothecin in 3% DMA or vehicle.

[0042]FIG. 9 illustrates survival curves from the first melanoma study.

[0043]FIG. 10 provides a time to ten-fold growth in tumor size analysisfor data from the first melanoma study.

[0044]FIG. 11 provides a time to ten-fold growth in tumor size analysisfor data from the second melanoma study.

[0045] FIGS. 12A-12D provide individual mouse data for the second 375melanoma study.

[0046]FIG. 12A provides individual mouse data for the first group fromthe second 375 melanoma study.

[0047]FIG. 12B provides individual mouse data for the second group fromthe second 375 melanoma study.

[0048]FIG. 12C provides individual mouse data for the third group fromthe second 375 melanoma study.

[0049]FIG. 12D provides individual mouse data for the fifth group fromthe second 375 melanoma study.

[0050]FIG. 13 illustrates the protocol design and study resultsevaluating IDD-P in a MX-1 human breast cancer xenograft study.

[0051]FIG. 14 illustrates the protocol design and study resultsevaluating IDD-P in a pan c-human pancreatic cancer xenograft study.

[0052]FIG. 15 illustrates the protocol design and study resultsevaluating IDD-P in a HT-29 human colon cancer xenograft study.

[0053]FIG. 16 illustrates the protocol design and study resultsevaluating IDD-P in a SKMES human lung cancer xenograft study.

DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention provides novel compositions that comprisecamptothecin compounds that include analogs, derivatives andpharmacologically active metabolites of 20(S)-camptothecin (collectivelyreferred to herein as “camptothecin compounds”) for the treatment ofneoplastic diseases or diseases associated with abnormal cellproliferation. The present invention also provides methods for treatingthese diseases through the delivery of the camptothecin compoundcomprising compositions of the present invention. The compositions aredesigned to be injectable and are preferably delivered intraveneously orintrathecally.

[0055] One feature of the present invention is that the camptothecincompound, especially a water-insoluble one such as 9-nitro-camptothecin,is solubilized and stabilized in an injectable formulation comprising anaqueous suspension of solid particles comprising the camptothecin, thesolid particles having mean diameters between about 0.05 μm and 10 μm,the particles coated with a 0.3 nm to 3.0 μm thick layer of amembrane-forming amphipathic lipid. Alternatively, the camptothecincompound, especially a water-insoluble one such as 9-nitro-camptothecin,is solubilized and stabilized in microdroplets having a mean diameterless than one micron, and optionally between 200 Angstroms and onemicron, the droplets comprising a substantially water-insoluble,pharmacologically acceptable liquid containing a camptothecin surroundedby a layer comprising a phospholipid.

[0056] Another feature of the present invention is that the camptothecincompound, especially a water-insoluble one such as 9-nitro-camptothecin,is stabile to steam sterilization in the injectable formulations of thepresent invention. In one embodiment, an aqueous dispersion of solidparticles is provided which comprises a camptothecin as solid particleshaving mean diameters between about 0.05 μm and 10 μm wherein the solidparticles are surface stabilized with at least one membrane-formingamphipathic lipid, preferably a phospholipid, and wherein the solidparticles are dispersed in a continuous phase of a carrier, the carrierbeing an aqueous solution of one or more pharmaceutically acceptabletonicity modifier agents. Alternatively, the camptothecin compound issolubilized and stabilized as a thermally sterilized (or sterilizable)injectable aqueous dispersion of microdroplets having a mean diameterless than 2 μm, and optionally between 200 Angstroms and 1 μm, themicrodroplets comprising a substantially water-insoluble,pharmaceutically acceptable lipophillic liquid containing thecamptothecin compound and at least one membrane-forming amphipathiclipid, preferably a phospholipid, wherein the microdroplets aresurrounded by a layer comprising the membrane-forming amphipathic lipid,and wherein the microdroplets are dispersed in a continuous phase of anaqueous carrier solution of one or more pharmaceutically acceptabletonicity modifier agents.

[0057] The inventors have discovered that significant tumor growthsuppression activity of camptothecins, particularly9-nitro-camptothecin, can be achieved when the formulations according tothe present invention are administered by injection, particularly by anintravenous route. Further, tumor growth suppression activity has beenshown for multiple different types of cancers, including melanoma,breast cancer, liver cancer and pancreatic cancer.

[0058] 1. Camptothecin Compounds

[0059] -“Camptothecin compound”, as it is referred to in the presentinvention, includes the plant alkaloid 20(S)-camptothecin, waterinsoluble or substantially water insoluble analogs, derivatives,prodrugs and pharmaceutically active metabolites of 20(S)-camptothecin.Examples of camptothecin derivatives include, but are not limited to,9-nitro-20(S)-camptothecin, 9-amino-20(S)-camptothecin,9-methyl-camptothecin, 9-chloro-camptothecin, 9-flouro-camptothecin,7-ethyl camptothecin, 10-methyl-camptothecin, 10-chloro-camptothecin,10-bromo-camptothecin, 10-fluoro-camptothecin, 9-methoxy-camptothecin,11-fluoro-camptothecin, 7-ethyl-10-hydroxy camptothecin,10,11-methylenedioxy camptothecin, and 10,11-ethylenedioxy camptothecin,and 7-(4-methylpiperazinomethylene)-10,11-methylenedioxy camptothecin.Prodrugs of camptothecin include, but are not limited to, esterifiedcamptothecin derivatives as described in U.S. Pat. No. 5,731,316, suchas camptothecin 20-O-propionate, camptothecin 20-O-butyrate,camptothecin 20-O-valerate, camptothecin 20-O-heptanoate, camptothecin20-O-nonanoate, camptothecin 20-O-crotonate, camptothecin20-O-2′,3′-epoxy-butyrate, nitro-camptothecin 20-O-acetate,nitro-camptothecin 20-O-propionate, and nitro-camptothecin20-O-butyrate.

[0060] In an embodiment, the camptothecin compound has poor watersolubility, for example, 10 μg/ml or less. Examples of camptothecincompounds with poor water solubility include, but are not limited to,9-nitro-20(S)-camptothecin, 9-amino-20(S)-camptothecin,7-ethyl-10-(4-(1-piperdino)-1-piperdino)-carbonyloxy-camptothecin,7-ethyl-10-hydroxy-20(S)-camptothecin,10,11-methylenedioxy-20(S)-camptothecin, 9-chloro-20(S)-camptothecin,9-bromo-20(S)-camptothecin, 9-hydroxy-20(S)-camptothecin, and11-hydroxy-20(S)-camptothecin. In a preferred embodiment, the modified20(S)-camptothecin is 9-nitro-20(S)-camptothecin.

[0061] Native, unsubstituted, the plant alkaloid camptothecin can beobtained by purification of the natural extract, or may be obtained fromthe Stehlin Foundation for Cancer Research (Houston, Tex.). Substitutedcamptothecins can be obtained using methods known in the literature, orcan be obtained from commercial suppliers. For example,9-nitro-camptothecin may be obtained from SuperGen, Inc. (San Ramon,Calif.), and 9-amino-camptothecin may be obtained from IdecPharmaceuticals (San Diego, Calif.). Camptothecin and various of itsanalogs and derivatives may also be obtained from standard fine chemicalsupply houses, such as Sigma Chemicals.

[0062] 2. Injectable Compositions of the Present Invention

[0063] The present invention relates to pharmaceutical compositionssuitable for use in delivering a camptothecin by injection. Thepharmaceutical compositions may be divided into two categories: (a)phospholipid stabilized particulate aqueous dispersion of a camptothecinthat is stable to sterilization, especially by autoclaving; and (b)phospholipid stabilized aqueous dispersion of microdroplet containing acamptothecin that is stable to sterilization, especially by autoclaving.Each composition type is described herein.

[0064] Pharmaceutical compositions according to the invention mayadditionally contain suitable amounts of pH buffering salts and pHadjusting agents such as sodium hydroxide and/or pharmaceuticallyacceptable acids. Phospholipids molecules can undergo hydrolysis at pHlower than 5 and higher than 9. Therefore, the pH of the aqueous carriermedium for the particulate or droplet dispersion is preferably adjustedto within this range prior to homogenization, and if necessaryreadjusted prior to sterilization particularly when autoclaving by steamis used.

[0065] It is further noted with regard to the formulations of thepresent invention that the formulations are preferably acidic, i.e.,have a pH less than 7 in order to stabilize the α-hydroxy lactone ringof camptothecins. In a more preferred embodiment, the formulations havea pH less than 6.5, more preferably less than 6. Optionally, the pH ofthe formulations are between 5 and 7, more preferably between 5 and 6.5and most preferably between 5 and 6.

[0066] All parts and percentages reported herein are by weight (w/w) orweight/volume (w/v) percentage, in which the weight or volume in thedenominator represents the total weight or volume of the system.Concentrations of water soluble constituents in aqueous solution (e.g.glucose) are given in millimolar concentration (mM=millimoles per liter)referred to the volume of water in the system. All temperatures arereported in degrees Celsius. Diameters or dimensions are given inmillimeters (mm=10⁻³ meters), micrometers (μm,=10⁻⁶ meters), nanometers(nm=10⁻⁹ meters) or Angstrom units (=0.1 nm). The compositions of theinvention can comprise, consist of, or consist essentially of thematerials set forth and the process or method can comprise, consist of,or consist essentially of the steps set forth with such materials.

[0067] A. Surface Stabilized Solid Particulate Dispersion of aCamptothecin of Micrometer to Submicrometer Dimensions

[0068] According to one pharmaceutical composition category according tothe present invention, the composition comprises a phospholipidstabilized particle dispersion of a camptothecin, preferably9-nitro-camptothecin. Procedures for making this category ofpharmaceutical composition, as well as varying embodiments, aredescribed in U.S. Pat. Nos. 5,091,187, 5,091,188, Re 35,338, 5,145,684and 5,494,683, which are each incorporated by reference.

[0069] In one embodiment according to this category, the pharmaceuticalcomposition comprises an aqueous suspension of solid particlescomprising a camptothecin, the solid particles having mean diametersbetween about 0.05 μm and 10 μm, the particles coated with a 0.3 nm to3.0 μm thick layer of a membrane-forming amphipathic lipid.

[0070] In another embodiment according to this category, thepharmaceutical composition of a camptothecin comprises an injectableaqueous dispersion of micrometer to submicrometer size solid particlessuitable for intravenous delivery, the dispersed solid particlescomprising a camptothecin, surface stabilized with at least onemembrane-forming amphipathic lipid, preferably a phospholipid and thesolid particle phase dispersed in a continuous phase of a carrieraqueous solution of pharmaceutically acceptable tonicity modifieragents. The dispersed solid particles are preferably stable to steamsterilization, also referred to herein as autoclaving.

[0071] The size of the dispersed camptothecin particles may becontrolled using low shear which produces relatively larger particlesand high shear which produces relatively smaller particles. Low shearmethods include Waring Blender, “high” and propeller homogenization androtating tube and plunger homogenizers. High shear methods include theFrench Pressure Cell or “French Press” (SLM Instruments, Urbana, Ill.),sonication (Heat Systems Co., Melville, N.Y.) and Microfluidization(Microfluidics Corp., Newton Mass.). The latter, which is described byMayhew et al. in Biochim. Biophys Acta 775:169-174, 1984, isparticularly well suited for commercial production. In addition, otherhigh energy processes providing the forces of attrition, impaction, highshear, antisolvent and solvent precipitation, rapid expansion of acompressed normal or supercritical solution and the like in the presenceof a membrane-forming amphipathic lipid can be used to produce thesestable sub-micrometer and micrometer size particle dispersions.

[0072] In a preferred embodiment, the camptothecin forms a solid core ofthe particle, and the solid core is stabilized by the membrane formingphospholipid. Alternatively, camptothecin molecules entrapped within orbelow lipid membrane bilayers or monolayers can form a coating overanother pharmaceutically-acceptable solid material comprising the coreof the particle which is stabilized by the membrane formingphospholipid. In either case the core material should be reduced to <10μm or submicron dimensions and dispersed in an aqueous medium as aparticle stabilized by a membrane forming phospholipid.

[0073] Particle size reduction can be accomplished by sonication orother high energy treatments involving high shear or forces of attritionor impaction in the presence of a membrane forming lipid, preferably oneor a combination of more than one phospholipids. Phospholipids (or othermembrane forming lipids) present during the high energy particle sizereduction step envelope the freshly generated micrometer tosubmicrometer size core material to stabilize the particle. Thus, arequirement for this process is that the phospholipids (or othermembrane forming lipids) and the core material be present togetherduring the sonication or other high-energy particle size reduction anddispersion process. The common aspect or all of these preparativemethods is that the molecules of the phospholipid (or other membraneforming lipids) must have direct access to the core material during theparticle size reduction process to form a primary coating on the core.

[0074] It is possible to increase the relative thickness or amount ofthe membrane forming material associated with the primary coating byadding additional phospholipid to the suspension after sonication orother application of high shear for example by suspending the addedphospholipid by homogenization at low shear (for example, using “highspeed homogenizers”, propeller homogenizers, Waring blender, or rotatingtube and plunger homogenizers).

[0075] The resulting particle should be steam sterilizable such that thesize of the particle does not substantially change as a result of beingsterilized. The size of the particle should also be stable over time andto changes in temperature.

[0076] In addition to making use of phospholipids and othermembrane-forming lipids as a particle enveloping and surface stabilizingmaterial, the present invention utilizes the membrane-forming lipids asmechanical buffers, organizers of aqueous volume and retardants ofrecrystallization of the drug. This is achieved by excess phospholipid(or other membrane forming lipids) in the form of unilamellar andmulti-lamellar vesicles. Unilamellar vesicles of the phospholipid (orother membrane forming lipids) are formed as the main byproduct of thehigh energy particle size reduction step. Their retention in thepreparation was found to improve the long-term stability of theformulation. Also, preformed multi-lamellar vesicles (made byhomogenization) or uni-lamellar vesicles can be added to the preparationto improve its stability or pharmacokinetics. The vesicles of thephospholipid (or other membrane forming lipids) may remain looselyattached to the micrometer to submicrometer size dispersed particle.These peripheral vesicles associate and dissociate continuously in thepreparation. Previous experimentation, described in U.S. Pat. No.5,091,188, has shown that these loosely attached vesicles can be removedby repeated centrifugation and resuspension of the preparation.

[0077] When a peripheral phospholipid is present at 20% (w/v), themajority of the aqueous volume of the preparation is enclosed withinphospholipid membranes. This serves as a topological barrier torecrystallization of the drug. Reformed crystals can not be larger thanthe diameter of the vesicles or distance between the vesicles.

[0078] In another aspect, when the camptothecin is entrapped between themembrane layers, it is necessary to provide another material to comprisethe particle core material of the particle. This can be anypharmaceutically-acceptable water-insoluble substance which shouldgenerally have a water solubility of <5 mg/ml at physiological pH(6.5-7.4). It can be selected from, but is not limited to, paraffin,tristearin, ethyl oleate, cetostearyl alcohol, cetyl alcohol, myristylalcohol, stearyl alcohol, petrolatum or biocompatible polymer. The corematerial can also be another pharmaceutically active agent. There aremany cases in which combination therapy with camptothecin is desirable.

[0079] Sonication or a high-energy particle size reduction step is mostconveniently carried out with the core material at concentrations of 25%(w/v) or less and the membrane-forming lipid at 0.5% or greater. It isalso convenient to add further core material and to repeat the process.With a single batch method or step-wise addition, final concentrationsof up to 30% core material can be achieved. The sonication or otherhigh-energy particle size reduction process results in a syringableaqueous dispersion of surface stabilized particles of predominantlysub-micrometer dimensions, with the particles exhibiting Brownianmotion. In some cases, over a period of 1-2 days to several weeks, thedispersions can settle creating a distinct sedimented zone in which theconcentration of core material is 20-60% (w/v). The final concentrationand volume are dependent on the choice of core material and upon thechoice of the concentration of phospholipid (or other membrane forminglipids) in the loosely attached peripheral domain.

[0080] In most preparations the sedimented material is resuspendablewith inversion or gentle agitation to give a homogeneous and syringablesuspension, even after a period of months. In preferred compositions ofthis invention, any sedimented material is resuspendable with inversionor gentle agitation to give a homogeneous and syringable suspension,even after a period of months.

[0081] The slow sedimentation process can be used to concentrate thepreparation. Removal of the volume above the sedimentation zone after1-2 days results in preparations in which the core material is at 20-60%(w/v). Long-term storage typically results in no further settling. Thepreparations remain homogeneous, syringable and pharmaceuticallyacceptable for many months. Microscopic examination of thesepreparations reveals distinct micron and sub-micron diameter particlesof core material. The volume between these is almost completely filledwith the primary enveloping layers and by phospholipid vesicles. Theparticles exhibit only restricted Brownian Motion. Under microscopicobservation they are not observed to change position in relation to eachother. They vibrate or “dance in place” about their central position.This partial restriction of motion is probably an important factor inthe long-term stability of the preparation.

[0082] Sterilized aqueous dispersions of camptothecin according to thepresent invention can be put into dry form by lyophilization to yield apowder which can be later reconstituted with a pharmaceuticallyacceptable fluid prior to use.

[0083] In a preferred embodiment, a preferred composition can beobtained according to this invention by first forming a solution bydissolving the water soluble components such as pH buffering agents andtonicity modifiers such as mannitol and/or trehalose in a requiredquantity of water. Mannitol or trehalose are known to modify osmoticpressure to provide osmolalities of the suspensions suitable forintravenous injection. A suitable osmolality for an injectableformulation of this invention is one for example that is substantiallyisotonic with blood. Preferably, a pharmaceutically acceptable pHbuffering agent is used. A particularly preferred pH buffering agent issodium acetate/acetic acid.

[0084] A pre-mix dispersion may be prepared under a nitrogen atmosphereby adding a required quantity of one or more membrane-formingamphipathic lipids, preferably one or more phospholipids such as LipoidE80, and a solid powder of 9-nitro-camptothecin to the aqueous solutionwhile mixing under high shear to mix the powder and the membrane-forminglipid in the presence of the carrier solution. The pH of the premix isadjusted with 1N NaOH or 20% acetic acid to about 5.5. The acidificationof the premix helps to reduce potential hydrolysis of the α-hydroxylactone ring of 9-nitro-camptothecin during this part of thepreparation.

[0085] The pre-mix dispersion is then subjected to high pressurehomogenization with cooling as needed, preferably under nitrogen andpreferably at pressures in the range of 10,000 psi to 25,000 psi for anumber of volume passes sufficient to achieve a desired particle sizedistribution. The homogenized suspension is then treated with one ormore tonicity modifiers such as mannitol or trehalose and water,buffered to about 5.5, filled into vials, stoppered under a nitrogenatmosphere, and autoclaved preferably using terminal steam sterilizationfor example above 100° C. at about 121° C. or hotter for about 15 to 30minutes or longer and then cooled.

[0086] The volume-weighted diameters and the particle size distributionof the resulting steam sterilized suspensions may be determined with aMalvern Mastersizer Microplus apparatus which utilizes a method based ondiffraction of light by the particulate suspension to provide a measureof particle size.

[0087] These suspensions can be further diluted for example with waterfor injection or other suitable sterile aqueous solution with lowosmolality to render them isotonic with blood and suitable forintravenous injection. Alternatively, these formulations can be dilutedwith a placebo that has the same composition as the formulation but doesnot contain the active drug and may be prepared by the same process thatis utilized to prepare the formulation containing active drug.

[0088] In another embodiment, this invention comprises an injectablepharmaceutical composition comprising a dispersion of micrometer tosubmicrometer size solid particles in an aqueous carrier solution of oneor more pharmaceutically acceptable tonicity modifier agents, theparticles comprising: a camptothecin drug, a first coating of not morethan 10% w/w of a substantially water-insoluble, pharmaceuticallyacceptable lipophilic agent in which the camptothecin drug is insolubleor poorly soluble, and a second coating of at least one membrane-formingsurface stabilizing amphipathic lipid, wherein the dispersion does notaggregate, flocculate, or agglomerate, and the particles do not grow insize above a volume weighted mean diameter of ten micrometers upon steamsterilization and storage prior to administration to a patient.

[0089] In another embodiment, this invention comprises an injectablepharmaceutical composition comprising a dispersion of micrometer tosubmicrometer size solid particles in an aqueous carrier solution of oneor more pharmaceutically acceptable tonicity modifier agents, theparticles comprising: a camptothecin drug, and a coating of at least onemembrane-forming surface stabilizing amphipathic lipid, wherein thedispersion does not aggregate, flocculate, or agglomerate, and theparticles do not grow in size above a volume weighted mean diameter often micrometers upon steam sterilization and storage prior toadministration to a patient.

[0090] In the above embodiments, the camptothecin drug can be present inamounts of up to about 25% w/w, preferably in amounts of from about0.05% w/w to about 5% w/w, more preferably in amounts of from about 0.1%w/w to about 1% w/w, and most preferably in an amount of about 0.2% w/w.

[0091] A. Selection of the Membrane-Forming Lipids for SurfaceStabilization

[0092] The primary requirement for lipids used in the compositions ofthe present invention is that the surface stabilizing lipid bemembrane-forming. This is satisfied by lipids which can, in the presenceof excess water, make bilayer structures of the type that iswell-documented for phospholipid vesicles or liposomes or which can, inthe presence of excess water, make monolayers by self aggregation. Thisrequirement is typically not satisfied by fats and oils such astriglycerides (vegetable oils, tristearin, “fats”). A secondaryrequirement is that the lipid not have a proclivity for converting intomicellar structures at the concentrations used in this invention. Thisexcludes phospholipids of short chain length (6 or less) orlyso-lecithin (containing a single fatty acyl chain). High stability ofthe membrane forming surface stabilizing material in membrane form isdesirable to keep the drug material from rearranging into macroscopiccrystals.

[0093] In addition to the requirement that the particle surfacestabilizing coating be membrane forming and not readily form micelles,the coating must also protect the camptothecin compound from interactionwith water that would lead to α-hydroxy lactone hydrolysis, especiallyduring formation of the particle, during the sterilization step in theformation of the dosage form, and during storage after the sterilizationstep and prior to administration of the camptothecin drug in the dosageforms of this invention.

[0094] The following list provides examples of membrane-forming lipidswhich may be used to form microparticles according to the presentinvention. This list is only intended to be illustrative.

[0095] 1. Primary Phospholipids

[0096] Lecithin

[0097] Phosphatidyl choline

[0098] Sphingomyelin

[0099] Synthetic zwitterionic phospholipids or phospholipid analogues

[0100] 2. Phospholipids Capable of Calcium-dependant Aggregation

[0101] Phosphatidic acid

[0102] Phosphatidyl serine

[0103] Phosphatidyl inositol

[0104] Cardiolipin (disphosphatidyl glycerol)

[0105] Phosphatidyl glycerol

[0106] 3. Phosphatidyl Ethanolamine

[0107] 4. Cholesterol and Steroids

[0108] 5. Semi-lipoidal Molecules

[0109] Stearylamine or other long-chained alkyl amines (includingprimary, secondary, tertiary or quaternary substituted).

[0110] Fatty acids

[0111] 6. Membrane-active Agents, Glycolipids and Glycoproteins

[0112] Nystatin

[0113] Amphotericin B

[0114] Gramicidin

[0115] Glycolipids or glycoproteins

[0116] 7. Mono-glycerides

[0117] 1-monopalmitoyl-(rac)-glycerol (Monopalmitin)

[0118] 1-monocaprylol-(rac)-glycerol (Monocaprylin)

[0119] 1-monooleoyl-(rac)-glycerol (C18:1, cis-9) (Monoolein)

[0120] 1-monostearyl-(rac)-glycerol (Monostearin)

[0121] 8. Commercially Available Membrane-Forming Lipids

[0122] Several forms of lecithin (egg lecithin (PfanstiehlLaboratories); bovine heart lecithin; soy bean lecithin; syntheticlecithin, preferably with 4 to 19 carbons (Supelco, Inc.); unsaturatedlecithins (dioleoyl, dilinoleoyl; beta oleoyl; alpha-palmito betaoleoyl; alpha palmitoyl beta linoleoyl and alpha oleoyl beta palmitoyl),diarachidonyl lecithin.

[0123] Phosphatidic acid available from egg or as synthetic compounds(dimyristoyl, dipalmitoyl or distearoyl, Calbiochem). Bovinephosphatidyl senna (Supelco or Calbiochem).

[0124] Phosphatidyl inositol available from plant (Supelco) or bovine(Calbiochem) sources.

[0125] Cardiolipin available (Supelco) from bovine or bacterial sources.

[0126] Phosphatidyl glycerol available from bacterial sources (Supelco)or as synthetic compounds (dimyristoyl or dipalmitoyl; Calbiochem).

[0127] Phosphatidyl ethanolamine available as egg, bacterial, bovine orplasmalogan (Supelco) or as synthetic compounds diotadecanoyl anddioleoyl analogues and dihexadecyl, dilauryl, dimyristoyl anddipalmitoyl (Supelco and Calbiochem).

[0128] Monoglycerides available from Sigma Chemical Co.(1-monopalmitoyl-(rac)-glycerol, monopalmitin;1-monocaprylol-(rac)-glycerol, monocaprylin; 1-monooleoyl-(rac)-glycerol(C18:1, cis-9), monoolein; 1-monostearyl-(rac)-glycerol, monostearin).

[0129] In preferred embodiments of this invention, the membrane-formingamphipathic lipid can be present in amounts of from 0.2% w/w to about 5%w/w, preferably in amounts of from 1% w/w to about 5% w/w, and morepreferably in amounts of about 4% w/w.

[0130] In another embodiment, compositions of this invention cancomprise a membrane forming lipid together with one or more than oneother surface active agent wherein combinations are chosen such thatthey fulfill the primary criteria of monolayer or bilayer membraneformation in presence of excess of water and the secondary criteria thatthe selected combination does not convert into micellar structures atthe concentrations used in this invention.

[0131] When compositions of this invention comprise a membrane forminglipid together with one or more than one other surface active agent, inaddition to the requirement that the particle surface stabilizingcoating formed in the presence of the other surface active agent bemembrane forming and not readily form micelles, the coating must alsoprotect the camptothecin compound from interaction with water that wouldlead to α-hydroxy lactone hydrolysis, especially during formation of theparticle, during the sterilization step in the formation of the dosageform, and during storage after the sterilization step and prior toadministration of the camptothecin drug in the dosage forms of thisinvention.

[0132] Surface active agents with high HLB numbers such asPEG-containing materials that are substantially more hydrophilic andeven soluble in water can potentially lead to enhanced access of waterto the surface of the particle when used to stabilize the particle. Theenhanced access of water to the camptothecin compound can lead toα-hydroxy lactone hydrolysis to form hydroxy acid compounds which aresubstantially less active in therapeutic applications such as thetreatment of cancer and other indications of the α-hydroxylactone-containing camptothecin compounds, and which are more toxic tohealthy tissue than the α-hydroxy lactone-containing camptothecincompounds. Thus, the amount of added other surface active compoundshould be less than the amount that would lead to hydrolysis of theα-hydroxy lactone ring during formation of the particle, duringsterilization, and during storage prior to use.

[0133] Representative surface active agents that can be present as longas the membrane forming property of the lipid is not compromised includethe following materials.

[0134] 1. Nonionic Surfactants

[0135] Examples include polyoxyethylene fatty alcohol ethers, sorbitanfatty acid esters, polyoxyethylene sorbitan fatty acid esters, sorbitanesters, glycerol esters such as and glycerol triacetate, polyethyleneglycols, cetyl alcohol, cetostearyl alcohol, stearyl alcohol,poloxamers, polaxamines, polyoxethylene castor oil derivatives, vitaminE or its derivatives, such as D-alpha-tocopheryl polyethylene glycol1000 succinate, PEG glyceryl fatty acid esters such as PEG-8 glycerylcaprylate/caprate, PEG-4 glyceryl caprylate/caprate, PEG-32 glyceryllaurate, PEG-6 glyceryl mono oleate, PEG-6 glyceryl linoleate, propyleneglycol mono- and di-fatty acid esters, such as propylene glycol laurate,propylene glycol caprylate/caprate; also diethylene glycol monoethylether, sorbitan fatty acid esters, monoglycerides and acetylatedmonoglycerides, e.g., glycerol monooleate, glycerol monostearate andmono- and di-acetylated monoglycerides, monoacetin, and diacetin;polyethylene glycol (PEG) such as PEG 300, PEG 400, PEG 600, PEG 1000,PEG 1500, PEG 3400.

[0136] 2. Anionic Surfactants

[0137] Examples include bile salts, potassium laurate, triethanolaminestearate, sodium lauryl sulfate, alkyl polyoxyethylene sulfates, sodiumalginate, dioctyl sodium sulfosuccinate, sodium carboxymethylcellulose,and calcium carboxymethylcellulose.

[0138] 3. Cationic Surfactants

[0139] Examples include quaternary ammonium compounds, benzalkoniumchloride, cetyltrimethylammonium bromide, andlauryldimethylbenzyl-ammonium chloride.

[0140] 4. Substituted Cellulose Derivatives

[0141] Examples include methylcellulose, hydroxycellulose, hydroxy

[0142] propylcellulose, hydroxy propylmethylcellulose, noncrystalline

[0143] cellulose, sodium carboxymethyl cellulose.

[0144] Optionally, a small quantity of one or more low-molecular weighthydrophilic substances, for example, monohydric or polyhydric alcohols,such as ethanol or glycols or glycerol may be also added in the surfacemodifiers as long as the membrane forming properties of the lipidsurface stabilizing agent are not compromised and α-hydroxy lactonehydrolysis in the camptothecin compound does not ensue. A detaileddescription of these surface modifying agents and lipophilic orhydrophobic media may be found in Remington's Pharmaceutical Sciences,18th Edition, 1990, Mack Publishing Co., PA; and Theory and Practice ofIndustrial Pharmacy, Lachman et al., 1986 and many monographs publishedthereafter.

[0145] It is possible to add other constituents to increase thestability of preferred embodiments of this invention or to modify therate of release of the camptothecin such as at a site of therapeuticaction. For example, pharmacologically-acceptable oils can be added atlow weight concentration to facilitate contact between the camptothecinmicrometer to submicrometer size particle surface and its protectivemembrane forming amphipathic lipid layer. It is necessary that the typeof oil and in weight concentration be chosen such that the crystallinedrug not be dissolved by the oil and that the coating by themembrane-forming lipid not be disrupted. These relationships can bedetermined empirically. Useful oils include, but are not limited to,vitamin E, isopropyl myristate, benzyl benzoate, oleyl alcohol, mineraloil, squalene and vegetable oil. The addition of these otherconstituents should not lead to enhancement in hydrolysis of theα-hydroxy lactone ring of the camptothecin compound in the compositionsof this invention during formation of the particles, duringsterilization, and during storage prior to use.

[0146] It is also possible to “precoat” the freshly formed micrometer tosubmicrometer size particles of camptothecin drugs by non-aqueous agentsthat are non-antigenic and compatible with surface stabilizing monolayeror bilayer membrane forming amphipathic agents. Useful agents includephospholipid-compatible, non-antigenic molecules which are solid at 37°C. Examples include paraffin, tristearin, ethyl oleate, cetostearylalcohol, cetyl alcohol, myristyl alcohol, stearyl alcohol andpetrolatum. For example, these materials can be incorporated onto thefreshly formed micrometer to submicrometer size particles ofcamptothecin drugs by sonication or shear. Stabilization can be achievedby adding surface stabilizing monolayer or bilayer membrane formingamphipathic agents during the particle size reduction process. It isdesirable to use low weight concentrations (less than 10%) such that thepayload is not degraded, and the rate or dissolution of the drug is notunduly impeded. Also, biodegradability may impose a further limitation.The use of these agents should not lead to enhancement in hydrolysis ofthe α-hydroxy lactone ring of the camptothecin compound in thecompositions of this invention during formation of the particles, duringsterilization, and during storage prior to use.

[0147] In one embodiment, preferred non-aqueous agents that can be usedto precoat freshly formed micrometer to submicrometer size particles ofcamptothecin drugs can be selected as individual compounds orcombination of compounds from the following group of compounds in whichthe camptothecin drug is either insoluble or poorly soluble, such asfrom the group of hydrophobic components such as triglycerides,diglycerides, monoglycerides, saturated or unsaturated free fatty acids,mixtures of saturated and unsaturated free fatty acids, and fatty acid,their esterification and their transesterification products obtained byreacting with alkanols, glycols, glycerol, or cholesterol. Examples ofsuch hydrophobic components include but are not limited to propyleneglycol dicaprylate/caprate, caprilic/capric triglyceride,caprylic/capric/linoleic triglyceride, e.g. synthetic medium chaintriglycerides having C₈₋₁₂ fatty acid chains or other derivatized(synthetic) triglycerides of the type known and commercially availableunder Miglyol 810, 812, 818, 829 and 840. Additional examples includeoils from biological sources such as vegetable oils and fish oils suchas soybean oil, almond oil, sunflower oil, olive oil, corn oil,polyunsaturated oils, saturated hydrogenated oils, fish oils, omega-3oils, omega-6 oils and C₁₂₋₁₈ fatty acid mono-, di- and triglyceridesprepared by individual admixing or as transesterification products ofvegetable oils or fish oils (such as soybean oil, almond oil, sunfloweroil, olive oil, corn oil, polyunsaturated oils, saturated hydrogenatedoils, omega-3 oils, omega-6 oils) with glycerol.

[0148] Additional non-aqueous agents that may be used for precoatinginclude pharmaceutically acceptable non-aqueous solvents in which thecamptothecin drug is either insoluble or poorly soluble. These solventsmay be selected from the classes of organic chemicals such as, but notlimited to, monohydric alcohols e.g., alkanols; dihydric alcohols e.g.,glycols; polyhydroxy compounds e.g., glycerin; aromatic esters, e.g.,benzyl benzoate, diethyl phthalate, propyl gallate; non-aromatic esterssuch as triacetin, diacetin, monoacetin, triethyl citrate; water solubleorganic solvents such as propylene carbonate and glycofurol, dimethylisosorbide, dimethyl isoidide, dimethyl isomannide, and otherpharmaceutically suitable hydrophobic compounds that remain in liquidstate at ambient temperature and pressure such as hydrofluorocarbonssuch as perflubron.

[0149] When preparing the composition for injection, particularly forintravenous delivery, the continuous phase preferably comprises anaqueous solution of tonicity modifiers, buffered to a pH below 7, morepreferably below 6. The tonicity modifier is preferably selected fromsodium chloride, glucose, mannitol, trehalose, glycerol, or otherpharmaceutically agent that renders the osmotic pressure of theformulation isotonic with blood. Alternatively, when a larger quantityof the tonicity modifier is used in the formulation, it can be dilutedprior to injection with a pharmaceutically acceptable diluent to renderthe mixture isotonic with blood.

[0150] Optionally, preservatives can be added. These include, but arenot limited to, benzalkonium chloride, propylparabem, butylparaben,chlorobutanol, benzyl alcohol, phenol, sodium benzoate, EDTA, etc. aslong as the membrane forming property of the lipid material is notcompromised. The addition of preservatives should not lead toenhancement in hydrolysis of the α-hydroxy lactone ring of thecamptothecin compound in the compositions of this invention duringformation of the particles, during sterilization, and during storageprior to use.

[0151] B. Surface Stabilized Microdroplet Dispersion of a Camptothecin

[0152] According to another pharmaceutical composition categoryaccording to the present invention, the composition comprises aphospholipid microdroplet suspension of a camptothecin, preferably9-nitro-camptothecin. Procedures for making this category ofpharmaceutical composition, as well as varying embodiments, aredescribed in U.S. Pat. Nos. 4,622,219 and 4,725,442, which are eachincorporated by reference.

[0153] In a preferred embodiment, the composition comprises aphospholipid stabilized aqueous dispersion of microdroplets ofpharmaceutically acceptable lipophillic liquid which sequesters acamptothecin, preferably 9-nitro-camptothecin. The composition ispreferably stabile to sterilization and is preferably sterilized, mostpreferably by steam sterilization.

[0154] In this aspect of the invention, the components of thedispersions should not permit hydrolysis of the α-hydroxy lactone ringof the camptothecin compound during formation of the microdroplets,during sterilization, and during storage prior to use.

[0155] In one embodiment according to this category, the pharmaceuticalcomposition comprises microdroplets having a mean diameter less than onemicron, and optionally between 200 Angstroms and one micron, thedroplets comprise a substantially water-insoluble, pharmacologicallyacceptable liquid containing a camptothecin surrounded by a layercomprising a phospholipid.

[0156] In a preferred aspect of this embodiment, the pharmaceuticalcomposition comprises a sterilized aqueous dispersion of microdroplets,preferably steam sterilized, suitable for injection, having a meandiameter less than one micron, and optionally between 200 Angstroms andone micron, the microdroplets comprise a substantially water-insoluble,pharmacologically acceptable lipophillic liquid vehicle, dissolvedwithin which a camptothecin drug and at least one membrane-formingamphipathic lipid, surrounded by a layer comprising at least onemembrane-forming amphipathic lipid, preferably a phospholipid, with themicrodroplets being dispersed in a continuous phase of a carrier aqueoussolution of pharmaceutically acceptable tonicity modifier agents.

[0157] As discussed elsewhere, the continuous aqueous phase of themicrodroplet dispersion composition is preferably acidic, i.e., have apH less than 7 in order to minimize hydrolysis of the α-hydroxy lactonering of camptothecins mediated by hydroxyl species such as can occur inbase catalyzed hydrolysis. In a more preferred embodiment, the aqueouscomponent of the composition has a pH less than 6.5, more preferablyless than 6. Optionally, the pH of the aqueous component of thecomposition is between 5 and 7, more preferably between 5 and 6.5 andmost preferably between 5 and 6.

[0158] Examples of pharmaceutically acceptable water-insoluble,lipophilic liquid vehicles include, but are not limited to alkanes,dialkyl ethers, long-chain esters, hydrophobic esters, biocompatiblesilicones, biocompatible high molecular weight fluorocarbons,oil-soluble vitamins and biocompatible volatile liquid anesthetics. Forintravenous injection use, preferred liquids include biocompatiblelong-chain esters, biocompatible hydrophobic esters, biocompatible highmolecular weight fluorocarbons, oil-soluble vitamins and biocompatibleliquid anesthetics.

[0159] The pharmaceutical composition is preferably an isotonicsolution. In this regard, the composition preferably further includestonicity modifiers, such as mannitol, glycerol, physiological saline,and/or trehalose.

[0160] The ratio of volume of the pharmacologically acceptable liquidcontaining a camptothecin to the weight of the phospholipid layer ispreferably at least 1.0 ml/g.

[0161] Some advantages believed to be provided by the microdropletcomposition include protection of the α-hydroxy lactone functional groupfrom hydrolysis in aqueous media, a relatively slow release of thecamptothecin compound to the tissues, and potential for a targeteddelivery to diseased tissues and cells by intelligent choice of the siteof injection with concomitant lowered metabolic degradation, first passeffects, and toxic side-effects in the liver and other organs.

[0162] A preferred method of preparing microdroplet dispersions is bysonication with a probe sonicator. Alternatively, microdroplets can beprepared in a bath sonicator. For small scale preparations a 1.0 cmdiameter test tube is suspended, with use of a test-tube clamp, in abath sonicator filled with water. The components of the microdroplet(lipophillic phase, phospholipid or membrane-forming amphipathic lipids,tonicity modifier, pH buffering agent, and drug to be included) arefirst grossly mixed by shaking, Vortex mixing, Polytron or othermethods. The homogenized suspension is then introduced into a bathsonicator and sonicated for 1-2 hours. If the preparation is to be doneon a large scale, it will be possible to omit the test tube andintroduce the components of the microdroplet dispersion directly intothe bath sonicator.

[0163] Microdroplet dispersions may also be produced by high intensitymechanical agitation. Useful methods include use of a Waring blender, aPolytron and high frequency shakers such as a commercial paint shaker.

[0164] An alternative method is a solvent dilution method. The desiredconstituents of the microdroplets are dissolved at high concentration inethanol or another oil- and water-miscible organic liquid. The ethanolsolution is rapidly diluted into an aqueous solution containing tonicitymodifier and pH buffering agents with vigorous mechanical agitation toinsure rapid mixing. The ethanol dissolves in the aqueous phase whilethe other constituents do not. The finely-dispersed constituentsspontaneously form microdroplet dispersions; the ethanol can beconveniently removed for example by dialysis or by means of vacuumevaporation or distillation.

[0165] Microdroplet dispersions can also be formed by a process similarto spray painting. The water-insoluble and oil-soluble constituents of amicrodroplet dispersion formulation of this invention are suspendedtogether and sucked into the intake of a commercial spray forming deviceand the resulting output spray can be directed through an aqueoussolution containing tonicity modifier(s) and pH buffering agents to forma microdroplet dispersion. The spray can be directed through the aqueoussolution from below the surface of the solution or from above thesurface of the aqueous solution, optionally through a gas such as afluorocarbon or sulfur hexafluoride or carbon dioxide optionallycontaining volatilized or atomized lipid molecules and optionally at atemperature above the temperature of the aqueous phase to potentiallymodify the surface properties of microdroplet through adsorption orabsorption of components of the gas.

[0166] In a preferred embodiment, a preferred composition can beobtained according to this invention by first forming an aqueous-phasein the form of a solution by dissolving the water soluble componentssuch as pH buffering agents and tonicity modifiers in a requiredquantity of water. Mannitol and trehalose are known to modify theosmotic pressure to provide osmolalities of the suspensions suitable forintravenous injection. A suitable osmolality is one for example that issubstantially isotonic with blood. Preferably, a pharmaceuticallyacceptable pH buffering agent is used. A particularly preferred pHbuffering agent is sodium acetate/acetic acid.

[0167] In parallel, an oil-phase may be prepared by dissolving desiredquantities of a camptothecin such as 9-nitro-camptothecin and one ormore membrane-forming amphipathic lipids such as one or morephospholipids for example Lipoid EPC and dimyristoylphosphatidylglycerol optionally with a membrane enhancing substance such ascholesterol in a required quantity of water-insoluble lipophilic liquidvehicle comprising a synthetic or a natural triglyceride or a mixturethereof, preferably in a desired quantity of a mixture of a medium chaintriglycerides and a vegetable oil such as soybean oil. Magnetic stirringis employed to facilitate dissolution. After complete dissolution of allcomponents, the solution is further stirred for at least an additionaltime to ensure complete solubilization and homogenization of thedissolved components. This oil phase preparation is carried out in aninert atmosphere such as a nitrogen atmosphere.

[0168] In a preferred embodiment, the lipophilic liquid vehicle orlipophilic agent is selected from the group consisting of vegetableoils, animal oils, synthetic oils, semi-synthetic oils, soybean oil,medium chain triglycerides, long chain triglycerides, triglycerides ofC8 to C12 saturated fatty acids, triglycerides of C14 to C22 saturatedfatty acids, triglycerides of C14 to C22 unsaturated fatty acids, andcombinations thereof. In addition, cholesterol can be used not only inthe lipophilic liquid vehicle and as a lipophilic agent of thisinvention, but also in the membrane-forming amphipathic lipid.

[0169] A pre-mix comprising a mixture of the oil-phase and water-phasemay be prepared by transferring the oil-phase under nitrogen pressureinto the water-phase and dispersing the mixture by rapid stirring undera nitrogen atmosphere. Sufficient quantity of dilute sodium hydroxidesolution or 20% acetic acid solution is added to adjust the pH to 7.5.

[0170] The pre-mix dispersion is then subjected to high-pressurehomogenization with cooling as needed, preferably under nitrogen andpreferably at a maximum pressure in the range of about 7,000 psi to8,000 psi for a number of volume passes sufficient to achieve a desireddroplet size distribution. The homogenized product is filled into vials,stoppered under a nitrogen atmosphere, and autoclaved preferably usingterminal steam sterilization for example at 121° C. or hotter for about15 to about 30 minutes or longer and then cooled.

[0171] The volume weighted diameters and particle size distribution ofthe resulting steam sterilized dispersion are determined with a Nicomp370 apparatus which utilizes quasi-elastic laser light scattering by thedispersed phase to provide accurate measure of very small particles ofas low as a about two micrometers or even smaller.

[0172] These dispersions can be further diluted for example withsuitable sterile aqueous solution with osmolality isotonic with bloodand suitable for injection. Alternatively, these formulations can bediluted with a placebo that has the same composition as the formulationbut does not contain the active drug and may be prepared by the sameprocess that is utilized to prepare the formulation containing activedrug.

[0173] Microdroplet dispersions according to the present invention maybe prepared from a wide variety of organic phases (i.e., thesubstantially water-insoluble, pharmacologically acceptable liquid)which, for convenience, may be considered by the following non-limitingtypes or categories: alkanes, natural and plant-derived “oils” (e.g.,olive oil and various vegetable oils), semisynthetic oils or fullysynthetic oils such as triglycerides of fatty acids, ethers (e.g.,dipropyl ether, n-dipropyl ether, dibutyl ether, and n-dibutyl ether),esters (e.g., a long-chain or hydrophobic ester), silicones, and highmolecular weight liquid fluorocarbons. For intravenous use, microdropletdispersions according to the present invention may be prepared from avariety of biocompatible organic phases including biocompatible naturaland plant-derived oils such as olive oil and various vegetable oils,biocompatible semisynthetic oils or biocompatible fully synthetic oilssuch as triglycerides of fatty acids, biocompatible fatty acid esters,and biocompatible high molecular weight liquid fluorocarbons.

[0174] Preferred agents suitable as substantially water-insoluble,pharmacologically acceptable lipophilic liquid vehicle can be anon-aqueous medium in which the camptothecin drug is either soluble orinsoluble or poorly soluble and is selected from the group ofhydrophobic components, either individually or in combination, such astriglycerides, diglycerides, monoglycerides, saturated or unsaturatedfree fatty acids, mixtures of saturated and unsaturated free fattyacids, and fatty acid, their esterification and theirtransesterification products obtained by reacting with alkanols,glycols, glycerol, or cholesterol. Examples of such hydrophobiccomponents include but are not limited to propylene glycoldicaprylate/caprate, caprilic/capric triglyceride,caprylic/capric/linoleic triglyceride, e.g. synthetic medium chaintriglycerides having C₈₋₁₂ fatty acid chains or other derivatized(synthetic) triglycerides of the type known and commercially availableunder Miglyol 810, 812, 818, 829 and 840. Additional examples includeoils from biological sources such as vegetable oils and fish oils suchas soybean oil, almond oil, sunflower oil, olive oil, corn oil,polyunsaturated oils, saturated hydrogenated oils, fish oils, omega-3oils, omega-6 oils and C₁₂₋₁₈ fatty acid mono-, di- and triglyceridesprepared by individual admixing or as transesterification products ofvegetable oils or fish oils (such as soybean oil, almond oil, sunfloweroil, olive oil, corn oil, polyunsaturated oils, saturated hydrogenatedoils, omega-3 oils, omega-6 oils) with glycerol.

[0175] In selecting water insoluble liquids of this invention, care mustbe taken to avoid liquids which can lead to facilitation of hydrolysisof the α-hydroxy lactone ring of the camptothecin compound duringpreparation of the microdroplet dispersion, during sterilization of themicrodroplet disperison, and during storage of the microdropletdispersion prior to use.

[0176] In another embodiment, additional non-aqueous media includepharmaceutically acceptable non-aqueous media or solvents in which thecamptothecin drug is either soluble or insoluble or poorly soluble.These solvents may be selected from classes of organic chemicals such asbut not limited to, monohydric alcohols e.g., alkanols; dihydricalcohols e.g., glycols; polyhydroxy compounds e.g., glycerin; aromaticesters, e.g., benzyl benzoate, diethyl phthalate, propyl gallate;non-aromatic esters such as triacetin, diacetin, monoacetin, triethylcitrate; water soluble organic solvents such as propylene carbonate andglycofurol, dimethyl isosorbide, dimethyl isoidide, dimethyl isomannide,and other pharmaceutically suitable hydrophobic compounds that remain inliquid state at ambient temperature and pressure such ashydrofluorocarbons such as perflubron. In selecting these non-aqueousmedia, care must be taken to avoid media which can lead to facilitationof hydrolysis of the α-hydroxy lactone ring of the camptothecin compoundduring preparation of the microdroplet dispersion, during sterilizationof the microdroplet disperison, and during storage of the microdropletdispersion prior to use. Compounds that readily irreversibly react withthe camptothecin α-hydroxy lactone to form an inactive camptothecincompound are also to be avoided unless used in substantially smallamounts so that the camptothecin compound remains substantially intactin the α-hydroxy lactone form

[0177] Optionally, small quantity of low-molecular weight lipid solublesubstances, for example, monohydric or polyhydric alcohols, such asethanol or glycols or glycerol may be also added in the non-aqueousmedium for the vehicle. In selecting these low molecular weightsubstances, care must be taken to avoid substances which can lead tofacilitation of hydrolysis of the α-hydroxy lactone ring of thecamptothecin compound during preparation of the microdroplet dispersion,during sterilization of the microdroplet disperison, and during storageof the microdroplet dispersion prior to use. Compounds that readilyirreversibly react with the camnptothecin α-hydroxy lactone to form aninactive camptothecin compound are also to be avoided unless used insubstantially small amounts so that the camptothecin compound remainssubstantially intact in the α-hydroxy lactone form.

[0178] Various membrane-forming amphipathic lipids described above foruse in preparing the surface stabilized micrometer to submicrometer sizesolid particulate dispersion of camnptothecin drugs are suitable for usein preparing the microdroplet dispersions of the present invention.Mixtures of two or more such lipids are useful to vary the surfaceproperties and reactivity. Examples of lipids that may be used, include,but are not limited to lecithin, phosphatidyicholine, sphingomyelin,phosphatidic acid, phosphatidyl serine, phosphatidyl inositol,cardiolipin (diphosphatidyl glycerol), phosphatidyl glycerol,phosphatidyl ethanolamine, positively or negatively chargedphospholipids, electroneutral phospholipids, natural phospholipids,semisynthetic phospholipids or fully synthetic phospholipids, saturatedphospholipids, unsaturated phospholipid, and combinations thereof;steroids such as cholesterol and estrogens (e.g., estirol, estrone,estradiol and diethylstilbestrol), androgens such as androstenedione andtestosterone, semi-lipoidal molecules which can incorporate into themonolayer and change the surface activity of the microdroplet, such asstearylamine or other long-chained alkyl amines which can be primary orsecondary, but preferably non reacting hindered or non nucleophilicamines such as hindered primary, secondary, tertiary or quaternarysubstituted, arachidonic acid or fatty acids, and membrane-active agentsincluding nystatin, amnphotericin B and gramicidin. In selecting thesesubstances, care must be taken to avoid substances which can lead tofacilitation of hydrolysis of the α-hydroxy lactone ring of thecamptothecin compound during preparation of the microdroplet dispersion,during sterilization of the microdroplet disperison, and during storageof the microdroplet dispersion prior to use. Compounds that readilyirreversibly react with the camptothecin α-hydroxy lactone to form aninactive camptothecin compound are also to be avoided unless used insubstantially small amounts so that the camptothecin compound remainssubstantially intact in the α-hydroxy lactone form.

[0179] In regard to lecithin, several forms may be used. For examplelecithin is available as egg or bovine heart lecithin (natural) or inseveral synthetic varieties which differ in chain length. These includechain lengths ranging from 4 to 19 carbons (Supelco, Inc.). It isbelieved that lecithins with chain lengths in the biological range(10-18 carbons) are useful in various applications. Specific examples oflecithins include dimyristoyl (14 carbons), and didodecanoyl (12carbons).

[0180] Unsaturated lecithins (dioeoyl), dilinoeoyl; alpha-palmito, betaoleoyl; alpha palmitoyl beta linoleoyl and alpha oleoyl beta palmitoyl)are also available. Diarachidonyl lecithin (highly unsaturated and aprostaglandin precursor) is also available, as is alpha palmito betamyristoyl (mixed unsaturated chains) lecithin. Phosphatidic acid isavailable from egg or as synthetic compounds (dimyristoyl, dipalmitoylor distearoyl, Calbiochem). Bovine phosphatidyl serine is available(Supelco or Calbiochem). Phosphatidyl inositol is available from plant(Supelco) or bovine (Calbiochem) sources. Cardiolipin is available(Supelco) from bovine or bacterial sources. Phosphatidyl glycerol isavailable from bacterial (Supelco) sources or as synthetic compounds(dimyristoyl or dipalmitoyl; Calbiochem). Phosphatidyl ethanolamine isavailable as egg, bacterial, bovine, or plasmalogen (Supelco) or assynthetic compounds dioctadecanoyl and dioleoyl analogues anddihexadecyl, dilauryl, dimyristoyl and dipalmitoyl (Supelco andCalbiochem). A number of natural and synthetic phospholipids useful inthis invention are also available from Avanti Polar Lipids and fromLipoid GmbII.

[0181] Preferred membrane forming lipids include natural and syntheticlipids such as hen egg-derived phospholipid (egg phospholipid andpurified egg phospholipid), soy phospholipid, dimyristoyl lecithin,didodecanoyl lecithin, dioeoyl lecithin, dilinoeoyl lecithin,alpha-palmito-beta-oleoyl lecithin, alpha-palmitoyl-beta-linoleoyllecithin, alpha-oleoyl-beta-palmitoyl lecithin, diarachidonyl lecithin,alpha-palmito-beta-myristoyl lecithin, dimyristoyl phosphatidic acid,dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid,phosphatidyl serine, phosphatidyl inositol, dimyristoyl phosphatidylglycerol or dipalmitoyl phosphatidyl glycerol, dioctadecanoylphosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine,dihexadecyl phosphatidyl ethanolamine, dilauryl phosphatidylethanolamine, dimyristoyl phosphatidyl ethanolamine, dipalmitoylphosphatidyl ethanolamine, Lipoid E80, Lipoid ES, Lipoid 90H, and Lipoid100H. A particularly preferred membrane forming lipid is Lipoid E80.

[0182] In one embodiment, the invention comprises an injectablepharmaceutical composition comprising a dispersion in an aqueous carriersolution of one or more pharmaceutically acceptable tonicity modifieragents of micrometer to submicrometer size spherical liquid droplets ofa substantially water-insoluble, pharmaceutically acceptable lipophilicliquid vehicle containing in a dissolved form a camptothecin drug and atleast one membrane-forming amphipathic lipid, the liquid droplets beingsurrounded by a layer comprising at least one membrane-formingamphipathic lipid, wherein upon steam sterilization and storage prior toadministration to a patient, the dispersion does not aggregate,flocculate, agglomerate, or coalesce, and the droplets do not grow insize above a volume weighted mean diameter of ten micrometers.

[0183] In another embodiment, the invention comprises an injectablepharmaceutical composition comprising an aqueous carrier solution of oneor more pharmaceutically acceptable tonicity modifier agents, saidsolution containing a dispersion of spherical liquid droplets of a firstsize distribution of a substantially water-insoluble, pharmaceuticallyacceptable lipophilic liquid vehicle, said vehicle containing solidparticles of a camptothecin drug of a second size distribution togetherwith the camptothecin drug in a dissolved form and at least onemembrane-forming amphipathic lipid, the liquid droplets being surroundedby a layer comprising at least one membrane-forming amphipathic lipid,wherein the first size distribution is in the range of submicrometer tomicrometers, and the second size distribution is smaller than the firstsize distribution, and wherein upon autoclave sterilization and storageprior to administration to a patient, the dispersion does not aggregate,flocculate, agglomerate, or coalesce, and the droplets do not grow insize above a volume weighted mean diameter of ten micrometers.

[0184] In these embodiments, the lipophilic liquid vehicle can bepresent in amounts up to about 40% w/w, preferably in amounts up toabout 25% w/w, and more preferably in amounts up to about 20% w/w. Inpreferred embodiments, the lipophilic liquid vehicle is present inamounts greater than 0.5% w/w.

[0185] In preferred embodiments of this invention, the ratio ofphospholipid to camptothecin compound in the suspension is less than150, preferably less than 100, more preferably less than 50, even morepreferably less than 20, yet even more preferably less than 10, morepreferably less than 5, and most preferably less than 1. In a preferredembodiment, when preparing the composition for injection, particularlyfor intravenous delivery, the continuous phase is an aqueous solution oftonicity modifiers, buffered to a pH below 7, more preferably below 6.The tonicity modifier is selected from sodium chloride, glucose,mannitol, trehalose, glycerol, or other pharmaceutically agent thatrenders the osmotic pressure of the formulation isotonic with blood.Alternatively, when a larger quantity of the tonicity modifier is usedin the formulation to produce a hypertonic dispersion prior to use, itcan be diluted prior to injection with a pharmaceutically acceptablediluent to render the mixture isotonic with blood.

[0186] Optionally, preservatives can be added. These include, but arenot limited to, benzalkonium chloride, propylparabem, butylparaben,chlorobutanol, benzyl alcohol, phenol, sodium benzoate, EDTA, etc. Inselecting these substances, care must be taken to avoid substances whichcan lead to facilitation of hydrolysis of the -hydroxy lactone ring ofthe camptothecin compound during preparation of the microdropletdispersion, during sterilization of the microdroplet disperison, andduring storage of the microdroplet dispersion prior to use. Compoundsthat readily irreversibly react with the camptothecin-hydroxy lactone toform an inactive camptothecin compound are also to be avoided unlessused in substantially small amounts so that the camptothecin compoundremains substantially intact in the α-hydroxy lactone form.

[0187] The type and amount of membrane forming amphipathic agents andother ingredients used is based on the relative solubility and/orpolarity of these ingredients, as well as their mutual compatibility.The formulation compositions may be optimized with respect to eachcomposition individually. Compositions in accordance with the presentinvention may include additional ingredients, for example, diluents orbulking agents, anti-oxidants, preserving agents, and pH bufferingagents. In selecting these substances, care must be taken to avoidsubstances which can lead to facilitation of hydrolysis of the α-hydroxylactone ring of the camptothecin compound during preparation of themicrodroplet dispersion, during sterilization of the microdropletdisperison, and during storage of the microdroplet dispersion prior touse. Compounds that readily irreversibly react with the camptothecinα-hydroxy lactone to form an inactive camptothecin compound are also tobe avoided unless used in substantially small amounts so that thecamptothecin compound remains substantially intact in the α-hydroxylactone form.

[0188] Without wishing to limit this invention by being bound by aparticular theory of its operation, it is thought that the combinationof membrane forming amphipathic surface active agents or lipophilic oilyvehicle and non-surface active water soluble agents perform severaluseful functions. These functions are believed to include suppressionthe process of Ostwald Ripening during the cooling cycle of the terminalsteam sterilization (vide infra) and therefore maintain the particlesize, increase the storage stability, minimize sedimentation, anddecrease the particle growth during lyophilization and reconstitution.

[0189] The association between the surface modifier and the drugparticles is believed to be enhanced such that the protectingenvironment around the particles is maintained over a wide range oftemperature and pressure as is prevalent during the terminal steamsterilization process and substantially minimizing hydrolysis of theα-hydroxy lactone ring during autoclaving.

[0190] The interface compatibility between water-insoluble disperseddrug particles or dispersed lipophillic droplet surface and thecontinuous aqueous medium is believed to be enhanced by the presentinvention.

[0191] The present invention is also believed to aid in orienting thesurface modifiers' hydrophilic portion preferentially into the aqueousphase while the lipophilic portion remains strongly adsorbed onto thesurface of the water-insoluble drug particle or the surface oflipophillic microdroplet as well as to enhance the stability of suchorientation.

[0192] It is further thought that the improved in-vivo efficacy ofcamptothecin drugs in the formulations of the present invention is atleast partially due to sequestering of the majority of the drugsubstance in the form of solid particle or in the dissolved state in anoily vehicle with the aid of combination of surface active agents orlipophilic oily vehicle and non-surface active water soluble agents insuch a way that the camptothecin drug molecules are not substantiallyavailable to high affinity protein binding sites that can destabilizethe active α-hydroxy lactone form of the drug within an in-vivoenvironment.

[0193] Emphasizing again that there is no intention of restricting thisinvention to any theory, cholesterol, triglycerides (in the form ofnatural or synthetic triglycerides) and phospholipids, that are presentin the preferred embodiments of this invention, may be required byrapidly proliferating neoplastic cells for their cellular functions. Thecompounds thus may serve to facilitate preferential uptake of thecomponents of these formulations by these cells.

[0194] It is also believed that the in-vivo efficacy of camptothecindrugs demonstrated herein arise from the combination of surface activeagents or lipophilic oily vehicle and non-surface active water solubleagents helping to preserve the camptothecin drug molecules in an activeα-hydroxy lactone form in therapeutically effective quantities in atherapeutically effective timeframe during their delivery to the targetsites of the neoplastic cells to cause lethal damage to these cells.

[0195] C. Sterilization of Camptothecin Formulations

[0196] Formulations of camptothecin and camptothecin derivatives such as9-nitro-camptothecin suitable for intravenous administration need to besterilizable. Prior to administration, the compositions of thisinvention must undergo a sterilization step. Two common methods forsterilization of formulations suitable for injection are describedherein.

[0197] In one scenario, a formulation can be generated under asepticconditions using components or mixtures of components that are eachsterilized prior to preparation of the final formulation and containerfilling such as filling of a vial and sealing or capping the file. Thismethod may be desirable when one or more components of the formulationis subject to chemical modification such as hydrolysis of a α-hydroxylactone ring or when the formulation such as a liposome is unstable tocommonly used methods of sterilization such as thermal autoclaving ortreatment with steam for a time sufficient to destroy any tracequantities of contaminating organism. However, this method is generallyexpensive to perform, requiring great care to exclude contaminants priorto fmal sealing of the dosage form, the creation and maintenance of anaseptic environment, and the manipulation of ingredients and processconditions under aseptic conditions to generate a desired sterileformulation.

[0198] It is more desirable to prepare a composition, seal it in a vial,and autoclave it such as with the use of steam applied to the vial abovethe boiling point of water such as at or above 121° C. for a time suchas about 15 to 20 minutes or longer to ensure the destruction of anytrace amount of contaminating organism. The sealed vial may then cooledand stored at a suitable temperature ready for use.

[0199] A preferred method of thermal sterilization of a composition ofthis invention comprises heating the composition in a vial with steam toat least 121° C. for at least 15 minutes.

[0200] Thermal sterilization of the composition post formulation issimpler to accomplish and relatively less expensive than one requiringpre-sterilization of all components and manipulation of ingredients in atotally aseptic environment. It is also a generally preferred method ofsterilization in the pharmaceutical industry. However, in an aqueousmedium labile chemical functional groups that may be present inconstituents of the formulation can potentially react with water underelevated temperature conditions. In addition, species that may bepresent in a formulation in an aqueous medium such as hydroxyl ions,protons, nucleophilic species such as carboxylate ions, metal ions andchelated metal ions even in trace quantities can potentially catalyzethe reaction with water of labile chemical functional groups that may bepresent. Such catalysts generally require the functional group to beaccessible to them, for example in the form of a part of a molecule thatis dissolved in a solvent or otherwise entropically free to react withwater. Examples of potentially labile functional groups includeα-hydroxy lactones (i.e., cyclic esters) such as the one present incamptothecins, which can hydrolyze in water to form alcohols andcarboxylic acids, certain labile carboxylic acid esters which canhydrolyze in water to form alcohols and carboxylic acids and/orcarboxylates especially in the presence of a catalyst, and phosphateesters which can hydrolyze in water to form alcohols and phosphoricacids and/or phosphates. Additionally, compositions containing smalldrug particles in the nanometer and micron size distribution range thatis acceptable for injection when in that size range can potentiallyincrease in size because of Ostwald ripening under stress conditionssuch as under mechanical stress by shaking, and under thermal stresssuch as by increasing and decreasing the temperature of the formulationor by applying temperature gradients to the formulation. In addition,suspensions of particles can also show time and/or temperature dependentinstability that results in irreversible agglomeration of particles togive effectively larger size distributions, and coagulation ofcomponents to give precipitates that cannot be resuspended with mildshaking or with gentle swirling. The micrometer to submicrometer sizeparticles of a particulate dispersion dosage forms may also suffer fromirreversible aggregation upon steam sterilization stress. Similarly, themicrometer to submicrometer size droplets of a microdroplet dispersiondosage forms may also suffer from irreversible coalescence upon steamsterilization stress. Furthermore, if a formulation is frozen and thenthawed, the stress of such a process may frequently lead to irreversibleprecipitation and coagulation of some or all of the components.

[0201] WO 99/61001, which is incorporated by reference in its entirety,describes processes for thermally sterilizing certain aqueoussuspensions of phospholipid stabilized particles. The processesdescribed in WO 99/61001 are particularly useful for sterilizingsuspensions of small particles after they are placed in vials and sealedprior to use. These processes may be used to heat sterilize the aqueousformulations according to the present invention.

[0202] As demonstrated in the examples herein, the heat sterilizedpharmaceutical compositions of the present invention where shown to bestable under various storage conditions that might be encountered aftersterilization during a storage time prior to administration. The heatsterilized pharmaceutical compositions of the present invention whereshown to be stable to freeze-thaw cycling, stable to thermal cyclingstress, and stable to mechanical stress, and stable to hydrolysis understeam sterilization conditions. It is anticipated that after thermalsterilization, the preferred compositions of this invention will bestable during a storage time prior to administration. The storage timecan be relatively short, i.e., a composition can be used immediatelyafter cooling to a temperature suitable for administration such as fromabout 5° C. to about 37° C. Preferably however, the storage time isrelatively long, i.e., a composition can be sterilized and then cooledto a suitable storage temperature such as from about 5° C. or less toabout room temperature such as from about 15° C. to about 25° C., for astorage time ranging up to at least one month, preferably up to at leastsix months, more preferably up to at least one year, and most preferablyup at least 2 years or more. Alternatively, a composition of thisinvention can be sterilized and then frozen during a first storage timesuch as any of those outlined above, then thawed and used during asecond storage time above the freezing point of the composition prior toadministration where the second storage time is at least one hour,preferably at least one day, more preferably at least one week, evenmore preferably at least one month, even more preferably at least sixmonths, yet even more preferably at least one year, and most preferablyat least two years.

[0203] 3. Administration of the Intravenous Formulations

[0204] The present invention also provides a method for treatingpatients suffering from abnormal cell proliferation such as cancer byusing the pharmaceutical composition described above.

[0205] The pharmaceutical compositions of the present invention, priorto administration, may be further diluted as desired by adding anaqueous solution, such as water, saline or other infusion fluid, andused for intravenous injection or infusion. For example, thepharmaceutical composition can be diluted with sterile water, normalsaline, D5W, Ringer's solution or other equivalent infusion liquids.Dilution of the composition preferably ranges from 1:10 to 1:500 v/v ofthe composition to the diluting infusion liquids. The dilution may alsobe appropriately adjusted according to the specific treatment schemesadopted by clinicians. The ratio of v/v in this context refers to theratio of the volume of the composition before dilution with the infusionfluids to the total volume of the pharmaceutical formulation followingdilution with the infusion fluid.

[0206] Prior to administration, the compositions of the presentinvention may contain varying amounts of a camptothecin compound. In apreferred embodiment, the concentration of the camptothecin compound inthe composition is greater or equal to about 1.0 mg/ml, and morepreferably greater or equal to about 2 mg/ml.

[0207] 4. Indications for Treatment with the Composition of the PresentInvention

[0208] Preferable indications that may be treated using the compositionsof the present invention include those involving undesirable oruncontrolled cell proliferation. Such indications include benign tumors,various types of cancers such as primary tumors and tumor metastasis,restenosis (e.g. coronary, carotid, and cerebral lesions), abnormalstimulation of endothelial cells (atherosclerosis), insults to bodytissue due to surgery, abnormal wound healing, abnormal angiogenesis,diseases that produce fibrosis of tissue, repetitive motion disorders,disorders of tissues that are not highly vascularized, and proliferativeresponses associated with organ transplants. It is noted, however, thatthe formulations of the present invention may be used to intraveneouslyor intrathecally deliver camptothecin for any indication for whichcamptothecin is useful.

[0209] Generally, cells in a benign tumor retain their differentiatedfeatures and do not divide in a completely uncontrolled manner. A benigntumor is usually localized and nonmetastatic. Specific types benigntumors that can be treated using the present invention includehemangiomas, hepatocellular adenoma, cavernous haemangioma, focalnodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma,bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas,teratomas, myxomas, nodular regenerative hyperplasia, trachomas andpyogenic granulomas.

[0210] In a malignant tumor cells become undifferentiated, do notrespond to the body's growth control signals, and multiply in anuncontrolled manner. The malignant tumor is invasive and capable ofspreading to distant sites (metastasizing). Malignant tumors aregenerally divided into two categories: primary and secondary. Primarytumors arise directly from the tissue in which they are found. Asecondary tumor, or metastasis, is a tumor which originated elsewhere inthe body but has now spread to a distant organ. The common routes formetastasis are direct growth into adjacent structures, spread throughthe vascular or lymphatic systems, and tracking along tissue planes andbody spaces (peritoneal fluid, cerebrospinal fluid, etc.)

[0211] Specific types of cancers or malignant tumors, either primary orsecondary, that can be treated using this invention include leukemia,breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer,lung cancer, brain cancer, cancer of the larynx, gallbladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cellcarcinoma of both ulcerating and papillary type, metastatic skincarcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma,myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet celltumor, primary brain tumor, acute and chronic lymphocytic andgranulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullarycarcinoma, pheochromocytoma, mucosal neuronms, intestinalganglloneuromas, hyperplastic corneal nerve tumor, marfanoid habitustumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignantmelanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

[0212] Treatment of abnormal cell proliferation due to insults to bodytissue during surgery may be possible for a variety of surgicalprocedures, including joint surgery, bowel surgery, and cheloidscarring. Diseases that produce fibrotic tissue include emphysema.Repetitive motion disorders that may be treated using the presentinvention include carpal tunnel syndrome. An example of cellproliferative disorders that may be treated using the invention is abone tumor.

[0213] The proliferative responses associated with organ transplantationthat may be treated using this invention include those proliferativeresponses contributing to potential organ rejections or associatedcomplications. Specifically, these proliferative responses may occurduring transplantation of the heart, lung, liver, kidney, and other bodyorgans or organ systems.

[0214] Abnormal angiogenesis that may be may be treated using thisinvention include those abnormal angiogenesis accompanying rheumatoidarthritis, ischemic-reperfusion related brain edema and injury, corticalischemia, ovarian hyperplasia and hypervascularity, (polycystic ovarysyndrom), endometriosis, psoriasis, diabetic retinopaphy, and otherocular angiogenic diseases such as retinopathy of prematurity(retrolental fibroplastic), macular degeneration, corneal graftrejection, neuroscular glaucoma and Oster Webber syndrome.

[0215] Diseases associated with abnormal angiogenesis require or inducevascular growth. For example, corneal angiogenesis involves threephases: a pre-vascular latent period, active neovascularization, andvascular maturation and regression. The identity and mechanim of variousangiogenic factors, including elements of the inflammatory response,such as leukocytes, platelets, cytokines, and eicosanoids, orunidentified plasma constituents have yet to be revealed.

[0216] In another embodiment of the present invention, a method isprovided for treating diseases associated with undesired anduncontrolled angiogenesis. The method comprises administering to apatient suffering from uncontrolled angiogenesis the pharmaceuticalcomposition of the present invention. The particular dosage of thecamptothecin compound requires to inhibit angiogenesis and/or angiogenicdiseases may depend on the severity of the condition, the route ofadministration, and related factors that can be decided by the attendingphysician. Generally, accepted and effective daily doses are the amountsufficient to effectively inhibit angiogenesis and/or angiogenicdiseases.

[0217] According to this embodiment, the composition of the presentinvention may be used to treat a variety of diseases associated withuncontrolled angiogenesis such as retinal/choroidal neuvascularizationand corneal neovascularization. Examples of retinal/choroidalneuvascularization include, but are not limited to, Bests diseases,myopia, optic pits, Stargarts diseases, Pagets disease, vein occlusion,artery occlusion, sickle cell anemia, sarcoid, syphilis, pseudoxanthomaelasticum carotid abostructive diseases, chronic uveitis/vitritis,mycobacterial infections, Lyme's disease, systemic lupus erythematosis,retinopathy of prematurity, Eales disease, diabetic retinopathy, maculardegeneration, Bechets diseases, infections causing a retinitis orchroiditis, presumed ocular histoplasmosis, pars planitis, chronicretinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma andpost-laser complications, diseases associated with rubesis(neovascularization of the angle) and diseases caused by the abnormalproliferation of fibrovascular or fibrous tissue including all forms ofproliferative vitreoretinopathy. Examples of corneal neuvascularizationinclude, but are not limited to, epidemic keratoconjunctivitis, VitaminA deficiency, contact lens overwear, atopic keratitis, superior limbickeratitis, pterygium keratitis sicca, sjogrens, acne rosacea,phylectenulosis, diabetic retinopathy, retinopathy of prematurity,correal graft rejection, Mooren ulcer, Terrien's marginal degeneration,marginal keratolysis, polyarteritis, Wegener sarcoidosis, Scleritis,periphigoid radial keratotomy, neovascular glaucoma and retrolentalfibroplasia, syphilis, Mycobacteria infections, lipid degeneration,chemical burns, bacterial ulcers, fungal ulcers, Herpes simplexinfections, Herpes zoster infections, protozoan infections and Kaposisarcoma.

[0218] In yet another embodiment of the present invention, a method isprovided for treating chronic inflammatory diseases associated withuncontrolled angiogenesis. The method comprises administering to apatient suffering from a chronic inflammatory disease associated withuncontrolled angiogenesis the pharmaceutical composition of the presentinvention. The chronic inflammation depends on continuous formation ofcapillary sprouts to maintain an influx of inflammatory cells. Theinflux and presence of the inflammatory cells produce granulomas andthus, maintains the chronic inflammatory state. Inhibition ofangiogenesis using the composition of the present invention may preventthe formation of the granulosmas, thereby alleviating the disease.Examples of chronic inflammatory disease include, but are not limitedto, inflammatory bowel diseases such as Crohn's disease and ulcerativecolitis, psoriasis, sarcoidois, and rhematoid arthritis.

[0219] Inflammatory bowel diseases such as Crohn's disease andulcerative colitis are characterized by chronic inflammation andangiogenesis at various sites in the gastrointestinal tract. Forexample, Crohn's disease occurs as a chronic transmural inflammatorydisease that most commonly affects the distal ileum and colon but mayalso occur in any part of the gastrointestinal tract from the mouth tothe anus and perianal area. Patients with Crohn's disease generally havechronic diarrhea associated with abdominal pain, fever, anorexia, weightloss and abdominal swelling. Ulcerative colitis is also a chronic,nonspecific, inflammatory and ulcerative disease arising in the colonicmucosa and is characterized by the presence of bloody diarrhea. Theseinflammatory bowel diseases are generally caused by chronicgranulomatous inflammation throughout the gastrointestinal tract,involving new capillary sprouts surrounded by a cylinder of inflammatorycells. Inhibition of angiogenesis by the composition of the presentinvention should inhibit the formation of the sprouts and prevent theformation of granulomas. The inflammatory bowel diseases also exhibitextra intestinal manifectations, such as skin lesions. Such lesions arecharacterized by inflammation and angiogenesis and can occur at manysites other the gastrointestinal tract. Inhibition of angiogenesis bythe composition of the present invention should reduce the influx ofinflammatory cells and prevent the lesion formation.

[0220] Sarcoidois, another chronic inflammatory disease, ischaracterized as a multisystem granulomatous disorder. The granulomas ofthis disease can form anywhere in the body and, thus, the symptomsdepend on the site of the granulomas and whether the disease is active.The granulomas are created by the angiogenic capillary sprouts providinga constant supply of inflammatory cells. By using the composition of thepresent invention to inhibit angionesis, such granulomas formation canbe inhibited. Psoriasis, also a chronic and recurrent inflammatorydisease, is characterized by papules and plaques of various sizes.Treatment using the composition of the present invention should preventthe formation of new blood vessels necessary to maintain thecharacteristic lesions and provide the patient relief from the symptoms.

[0221] Rheumatoid arthritis (RA) is also a chronic inflammatory diseasecharacterized by non-specific inflammation of the peripheral joints. Itis believed that the blood vessels in the synovial lining of the jointsundergo angiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen species that leadto pannus growth and cartilage destruction. The factors involved inangiogenesis may actively contribute to, and help maintain, thechronically inflamed state of rheumatoid arthritis. Treatment using thecomposition of the present invention alone or in conjunction with otheranti-RA agents should prevent the formation of new blood vesselsnecessary to maintain the chronic inflammation and provide the RApatient relief from the symptoms.

[0222] In a preferred embodiment, this invention comprises a method oftreatment of neoplastic cells or rapidly proliferating cells in apatient comprising administering by intravenous injection atherapeutically effective amount of an injectable pharmaceuticalcomposition comprising a dispersion of micrometer to submicrometer sizesolid particles in an aqueous carrier solution of one or morepharmaceutically acceptable tonicity modifier agents, the particlescomprising:

[0223] a camptothecin drug,

[0224] a first coating of not more than 10% w/w of a substantiallywater-insoluble,

[0225] pharmaceutically acceptable lipophilic agent in which thecamptothecin drug is

[0226] insoluble or poorly soluble, and

[0227] a second coating of at least one membrane-forming surfacestabilizing amphipathic lipid,

[0228] wherein the dispersion does not aggregate, flocculate, oragglomerate, and the particles do not grow in size above a volumeweighted mean diameter of 10 μm upon steam sterilization and storageprior to administration to a patient.

[0229] In another preferred embodiment, this invention comprises amethod of treatment of neoplastic cells or rapidly proliferating cellsin a patient comprising administering by intravenous injection atherapeutically effective amount of an injectable pharmaceuticalcomposition comprising a dispersion of micrometer to submicrometer sizesolid particles in an aqueous carrier solution of one or morepharmaceutically acceptable tonicity modifier agents, the particlescomprising:

[0230] a camptothecin drug, and

[0231] a coating of at least one membrane-forming surface stabilizingamphipathic lipid, wherein the dispersion does not aggregate,flocculate, or agglomerate, and the particles do not grow in size abovea volume weighted mean diameter of 10 μm upon steam sterilization andstorage prior to administration to a patient.

[0232] In another preferred embodiment, this invention comprises amethod of treatment of neoplastic cells or rapidly proliferating cellsin a patient comprising administering by intravenous injection atherapeutically effective amount of an injectable pharmaceuticalcomposition comprising a dispersion in an aqueous carrier solution ofone or more pharmaceutically acceptable tonicity modifier agents ofmicrometer to submicrometer size spherical liquid droplets of asubstantially water-insoluble, pharmaceutically acceptable lipophilicliquid vehicle containing in a dissolved form a camptothecin drug and atleast one membrane-forming amphipathic lipid, the liquid droplets beingsurrounded by a layer comprising at least one membrane-formingamphipathic lipid, wherein upon steam sterilization and storage prior toadministration to a patient, the dispersion does not aggregate,flocculate, agglomerate, or coalesce, and the droplets do not grow insize above a volume weighted mean diameter of 10 μm.

[0233] In another preferred embodiment, this invention comprises amethod of treatment of neoplastic cells or rapidly proliferating cellsin a patient comprising administering by intravenous injection atherapeutically effective amount of an injectable pharmaceuticalcomposition comprising an aqueous carrier solution of one or morepharmaceutically acceptable tonicity modifier agents, said solutioncontaining a dispersion of spherical liquid droplets of a first sizedistribution of a substantially water-insoluble, pharmaceuticallyacceptable lipophilic liquid vehicle, said vehicle containing solidparticles of a camptothecin drug of a second size distribution togetherwith the camptothecin drug in a dissolved form and at least onemembrane-forming amphipathic lipid, the liquid droplets being surroundedby a layer comprising at least one membrane-forming amphipathic lipid,wherein the first size distribution is in the range of submicrometer tomicrometers, and the second size distribution is smaller than the firstsize distribution, and wherein upon autoclave sterilization and storageprior to administration to a patient, the dispersion does not aggregate,flocculate, agglomerate, or coalesce, and the droplets do not grow insize above a volume weighted mean diameter of 10 μm.

[0234] In another preferred embodiment, this invention comprises amethod for administering a camptothecin to a patient comprisingintravenously administering to a patient a pharmaceutical compositionsuitable for intravenous administration to a mammal comprising anaqueous suspension of particles having mean diameters between about 0.05μm and 10 μm, the particles comprising a core containing a camptothecincompound having an intact lactone ring, wherein the core of eachparticle is stabilized against an increase in particle size of more than100% during a sterilization step and during a storage time by amembrane-forming amphiphatic lipid coating on the surface of the coreand by one or more tonicity modifying agents, wherein the ratio of lipidto the camptothecin compound is less than about 150:1 moles:mole, andwherein the coating substantially protects the lactone ring of thecamptothecin compound from interaction with water to prevent hydrolysisduring formation of the particle, during sterilization of the particlein the suspension, and during storage of the particle in the suspensionprior to administration.

[0235] In another preferred embodiment, this invention comprises amethod for administering a camptothecin to a patient comprisingintravenously administering to a patient a pharmaceutical compositionsuitable for intravenous administration to a mammal comprising anaqueous suspension of particles having mean diameters between about 0.05μm and 10 μm, the particles comprising a solid core containing acamptothecin compound having an intact lactone ring, wherein the core ofeach particle is stabilized against an increase in particle size of morethan 100% during a sterilization step and during a storage time by amembrane-forming amphiphatic lipid coating on the surface of the coreand by one or more tonicity modifying agents, wherein the ratio of lipidto the camptothecin compound is less than about 150:1 moles:mole, andwherein the coating substantially protects the lactone ring of thecamptothecin compound from interaction with water to prevent hydrolysisduring formation of the particle, during sterilization of the particlein the suspension, and during storage of the particle in the suspensionprior to administration.

[0236] In another preferred embodiment, this invention comprises amethod for administering a camptothecin to a patient comprisingintravenously administering to a patient a pharmaceutical compositionsuitable for intravenous administration to a mammal comprising anaqueous suspension of particles having mean diameters between about 0.05μm and 10 μm, the particles comprising a solid core of a camptothecincompound having an intact lactone ring, wherein the core of eachparticle is stabilized against an increase in particle size of more than100% during a sterilization step and during a storage time by amembrane-forming amphiphatic lipid coating on the surface of the coreand by one or more tonicity modifying agents, wherein the ratio of lipidto the camptothecin compound is less than about 150:1 moles: mole, andwherein the coating substantially protects the lactone ring of thecamptothecin compound from interaction with water to prevent hydrolysisduring formation of the particle, during sterilization of the particlein the suspension, and during storage of the particle in the suspensionprior to administration.

[0237] In another preferred embodiment, this invention comprises amethod for administering a camptothecin to a patient comprisingintravenously administering to a patient a pharmaceutical compositionsuitable for intravenous administration to a mammal comprising anaqueous suspension of particles having mean diameters between about 0.05μm and 10 μm, the particles comprising a core containing a camptothecincompound having an intact lactone ring, wherein the core of eachparticle is a liquid stabilized against an increase in particle size ofmore than 100% during a sterilization step and during a storage time bya membrane-forming amphiphatic lipid coating on the surface of the coreand by one or more tonicity modifying agents, wherein the ratio of lipidto the camptothecin compound is less than about 150:1 moles:mole, andwherein the coating substantially protects the lactone ring of thecamptothecin compound from interaction with water to prevent hydrolysisduring formation of the particle, during sterilization of the particlein the suspension, and during storage of the particle in the suspensionprior to administration.

EXAMPLES

[0238] The following examples set forth methods of preparingcompositions according to the present invention, studies regarding theirstability, as well as studies regarding their tumor growth suppressionfor different cancer types, specifically melanoma, breast cancer, lungcancer, and pancreatic cancer. In each study, a high level of tumorgrowth suppression was achieved using a formulation according to thepresent invention.

[0239] 1. Phospholipid Stabilized Solid Particulate Dispersion of9-Nitro-Camptothecin of Micrometer to Submicrometer Dimensions

[0240] In order to illustrate how one might prepare micrometer tosubmicrometer size particle dispersions of 9-nitro-camptothecin inaqueous injectable medium according to the present invention, thefollowing example describes the preparation of several such dispersions.One of the dispersions described herein (formulation 1-F, also referredto as IDD-P) was employed in the various tumor growth suppressionstudies described herein.

[0241] Aqueous dispersions of micrometer to submicrometer size particlesof 9-nitro-camptothecin containing the ingredients and amounts listed inFIG. 1 were prepared by adding a phospholipid and 9-nitro-camptothecinto an aqueous solution containing the remaining ingredients and stirringthe mixture under high shear to form a pre-mix dispersion under anitrogen atmosphere. The pH of the premix was adjusted with 1N NaOH or20% acetic acid to about 5.5. The acidification of the premix wasperformed in order to prevent hydrolysis of the α-hydroxy lactone ringof 9-nitro-camptothecin.

[0242] The pre-mix dispersion was then subjected to high pressurehomogenization under nitrogen at pressures in the range of 10,000 psi to25,000 psi for a number of volume passes sufficient to achieve thedesired particle size distribution by recirculating the process fluidthrough an Avestin Emulsiflex C5 homogenizer, Avestin Inc., ON, Canada.One volume pass is equal to the volume of the starting process fluidthat is pumped through the homogenizer's size reduction valve. Thematerial exiting the size reduction valve is continuously recirculatedto the homogenizer's inlet reservoir for reprocessing. During thehomogenization and particle size reduction process, the process fluidwas cooled by passing through a heat exchanger set to approximately 10°C. to maintain the dispersion at a temperature below 45° C. at the exitof the size reduction valve and elsewhere in the process fluid loop. Thehomogenized process fluid was then collected in a clean vessel atambient temperature under nitrogen atmosphere, and diluted with anaqueous solution containing pH buffering agents (sodium acetate andacetic acid) and tonicity modifier (either mannitol or trehalose) togive a fmal concentration of sodium acetate of 2 mM, and that of theother ingredients shown in FIG. 1.

[0243]FIG. 1 also summarizes representative sample compositions andobservations. For example, the amounts of 9-nitro-camptothecin (9NCA),phospholipid (Lipoid E80; employed as a surface modifier), andpolyhydroxy compounds (mannitol (MAN) and trehalose (TRE); useful astonicity modifying agents) are shown in the figure. Representativephysical attributes such as particle sizes of the formulations are alsoshown in the figure.

[0244] The pH of the diluted product was measured and adjusted to 5.5 asnecessary. The product was then filled into borosilicate USP Type Iglass vials. These vials were sealed under a nitrogen atmosphere withPTFE gray butyl stoppers and subjected to terminal steam sterilizationat 121° C. for 15 to 30 minutes. The volume-weighted diameters of theresulting suspensions, also reported in FIG. 1, were determined with aMalvern Mastersizer Microplus apparatus which utilizes a method based ondiffraction of light by the particulate suspension to provide a measureof particle size.

[0245] The steam sterilized suspensions were found to have particle sizedistributions that contained no particles greater than 7 μm. Suspensionsof particles in this size range are considered acceptable forintravenous injection into mammals, including human subjects.Formulations of identification 1-A, 1-B, and 1-C contain mannitol thatmodifies the osmotic pressure of the suspensions to provide osmolalitiesof the suspensions suitable for intravenous injection. A suitableosmolality is one for example that is substantially isotonic with blood.Formulations of identification 1-D, 1-Ea, 1-Eb, 1-Ec, 1-F and 1-Vcontain trehalose as the osmotic pressure modifier. These suspensionscan be diluted for example with water for injection or other suitablesterile aqueous solution with low osmolality to render them isotonicwith blood and suitable for intravenous injection.

[0246] The formulation identified as 1-V does not contain any activedrug and may be used as a placebo. This formulation can also be used asa diluent for the formulations containing the active drug in doseranging studies.

[0247] A. Stability of Sterile Suspensions of 9-nitro-camptothecinParticles Under Freeze-thaw Stress

[0248] The formulation identified as 1-F in FIG. 1 was used in thefollowing freeze/thaw stress experiment. This same formulation isidentified as IDD-P herein and was used in the below described tumorgrowth suppression studies.

[0249] A subset of vials containing the 1-F formulation was cooled in afreezer to approximately −20° C. for at least 6 hours to freeze thesuspensions contained therein. The frozen samples were then thawed byplacing the vials at room temperature for 0.5-1 hour. Particle sizedistribution of one of the thawed samples was measured by the methodmentioned above, and the appearance of the thawed sample was recorded.The remaining sample contained in the opened vial used for thesemeasurements was then discarded. The remaining vials of this set wereused for repeated freeze/thaw cycles similar to the above.

[0250] The formulation 1-F displayed very good particle size stabilityunder the stress of repeated freeze/thaw conditions. For example, theinitial volume weighted mean particle diameter of this formulation was1.29 μm and 99.9% of the particles were of a size less than 2.8 μm. Evenafter three cycles of the above freeze/thaw stress conditions, thevolume weighted mean particle diameter remained substantially unchangedat 1.28 μm and 99.9% of the particles were of a size less than 2.99 μm,substantially unchanged from the original measurement. This experimentdemonstrates the robustness of the formulation against the freeze/thawstress.

[0251] B. Stability of Sterile Suspensions of 9-nitro-camptothecinParticles Under Thermal Cycling Stress

[0252] The formulation identified as 1-F in FIG. 1 was also used in thefollowing thermal cycling stress experiment. In this experiment, athermal cycling stress sequence was applied to a subset of vialscontaining the 1-F formulation by storing the set for approximately 24hours in a refrigerator at about 4° C. and then in an incubator at about40° C. for approximately 24 hours. The refrigeration/warming cycle wasthen repeated for three cycles. The particle size of a thermallystressed suspension was measured and its appearance noted at the end ofeach cycle. After each particle size measurement, the vial used formeasurement was discarded.

[0253] The suspensions demonstrated very good particle size distributionstability and appearance after three cycles of thermal cycling stress.After three thermal cycles the volume weighted mean particle diameter ofthis formulation was substantially unchanged, increasing from an initialvalue of 1.29 μm to only 1.33 μm. In addition, 99.9% of the particlesremained below a size less than 3.44 μm after three thermal cycles. Thisdemonstrates the robustness of the formulation against the thermalcycling stress generated by repeated transfer or cycling of theformulation between 4° C. and 40° C.

[0254] C. Stability of Sterile Suspensions of 9-nitro-camptothecinParticles Under Shaking-standing Stress

[0255] The formulation identified as 1-F in FIG. 1 was also used in thefollowing shaking-standing stress experiment. In this example, a subsetof vials containing the 1-F formulation was placed on an orbital shakerwith each vial of the set being horizontal. The vials were shaken atapproximately 100 rpm. One vial was removed from the shaker daily forobservation of the appearance of the suspension and for particle sizemeasurement. The volume weighted mean particle size distribution and the99.9 percentile size of the suspension did not change significantly onshaking for up to 3 days after which the study was terminated. Afterthree days of shaking the volume weighted mean particle size remained1.28 μm and its 99.9 percentile remained under 3.02 μm.

[0256] D. Stability of Sterile Suspensions of 9-nitro-camptothecinParticles Under Different Temperatures for 170 Days

[0257] The formulation identified as 1-F in FIG. 1 was also used in thefollowing temperature stability experiment. Three sets of vialscontaining the sterilized formulation were placed in incubators held at4° C., 25° C., and 40° C. A vial was removed from the incubator forobservation of the appearance and particle sizing. FIG. 2 summarizes thevolume weighted mean particle size and 99.9 percentile data for thetemperature stability study. The volume weighted mean particle size ofthe suspensions did not change significantly on storage for up to 170days on storage at 4° C., 25° C., and 40° C. The 99.9 percentile sizedid not substantially change on storage for up to 170 days on storage at4° C. and 25° C.

[0258] 2. Method for Preparing Droplet Suspension of9-Nitro-camptothecin

[0259] In order to illustrate how to prepare phospholipid dropletsuspensions of 9-nitro-camptothecin according to the present invention,the following example describes the preparation of a formulation of9-nitro-camptothecin referred to herein as “IDD-D”. The IDD-Dformulation is employed in the various tumor growth suppression studiesdescribed herein.

[0260] First, an oil phase was prepared by dissolving 0.4 g of9-nitro-camptothecin, 40.0 g of Lipoid EPC (purified egg phosphatidylcholine, Lipoid GmbH, Ludwigshafen Germany), 20.0 g of cholesterol(Sigma, St. Louis, Mo.), and 4.0 g of dimyristoylphosphatidyl glycerol(Avanti Polar Lipids, Alabaster, Ala.) in a mixture of 200 g of a mediumchain triglyceride (Crodamol GTCC PN, Caprylic/Capric Triglyceride,Croda Inc., Parsippany, N.J.) and 200 g of soybean oil (Spectrum QualityProducts Inc., New Brunswick, N.J.) with magnetic stirring. Aftercomplete dissolution of all components, the solution was stirred for anadditional 10 min. A water phase was prepared by dissolving 80.0 g ofmannitol (J. T. Baker, Phillipsburg, N.J.) in 1455.6 g of water and thesolution was then purged with nitrogen.

[0261] An IDD-D premix comprising a mixture of the two solutions wasprepared by transferring the oil phase under nitrogen pressure into thewater-phase and dispersing the mixture by rapid stirring under anitrogen cover. Sufficient quantity of 1N NaOH or 20% acetic acid wasadded to adjust the pH to 7.5.

[0262] The premix was homogenized at 7,000-8,000 psig using aMicrofluidizer 110S (Microfluidics, Newton, Mass.) under a nitrogencover. The product was recirculated into the Microfluidizer via a heatexchanger cooled with a chiller set at 15-20° C. The homogenized productwas collected and filled into 10 mL USP Type I borosilicate clear glassvials under a nitrogen atmosphere using 10.2-10.5 g of product per vial.A TEFLON® faced gray butyl stopper was applied to each vial which wasthen further sealed with an aluminum flip-off seal. The sealed vials andtheir contents were steam sterilized by autoclaving at 121° C. for 20min with the vials maintained in a vertical position, and thenquench-cooled with about 10-15° C. water for 10 min. The steamsterilized product was characterized by pH determination and particlesizing (Nicomp 370; Particle Sizing Systems Inc., Santa Barbara, Calif.This equipment uses quasi-elastic laser light scattering for particlesize determination). The volume weighted particle size of the steamsterilized product, referred to herein as IDD-D, was 0.2 μm, and 99.9percent of the particle population was smaller than about 0.34 μm. Thesuspension had a pH of 7.0 after steam sterilization.

[0263] D. Stability of 9-nitro-camptothecin Suspension in IDD-D

[0264] The suspension formed according to this example was used forevaluating stability against the stress conditions detailed in theprevious examples, namely, storage at 2-8° C., 20° C., and 40° C.,thermal cycling between 4° C. and 40° C., and shaking at ambienttemperature. FIG. 3 summarizes the particle size stability data fromthese stability studies. Overall, the formulation displayed very goodparticle size stability under the different potentially destabilizingstress conditions.

[0265] 3. Melanoma Xenoeraft Model Study 1

[0266] The following study evaluates two intravenous formulationsaccording to the present invention against human tumor xenografts inathymic nude mice. The study used the A375 human melanoma xenograftmodel to compare the in vivo antitumor efficacy of I.V.9-nitro-camptothecin, in vehicles containing IDD-P which provides aparticulate suspension of 9-nitro-camptothecin, and IDD-D which providesa droplet suspension of 9-nitro-camptothecin, to the efficacy of oral9-nitro-camptothecin and intraperitoneal CAMPTOSAR®, HYCAMTIN®, andDTIC®. Examples 1 and 2 describe the compositions of IDD-P (FIG. 1;formulation 1-F) and IDD-D respectively.

[0267] In order to assess antitumor efficacy, groups of athymic nudemice (n=10) bearing ˜72-mg A375 melanomas were treated intravenously onthe 5/2/5 schedule with 9-nitro-camptothecin in IDD-P at 3.0 mg/kg or1.5 mg/kg and 9-nitro-camptothecin in IDD-D at 2.0 mg/kg or 1.0 mg/kg.

[0268] The 3.0 mg/kg doses of 9-nitro-camptothecin in IDD-P producedfive 62-day survivors, including one complete regression (CR) and threepartial regression (PR) responses. It is noted that a high 30% toxicdeath rate was observed.

[0269] The 1.5 mg/kg doses of 9-nitro-camptothecin in IDD-P produced twoCR and 1 SD/PD responses, no toxic deaths, and a mean day of survival(MDS) of 50.7 days (n=7), compared to the MDS of 29.2 days for thevehicle control group (n-10).

[0270] 9-nitro-camptothecin in IDD-D at 2 mg/kg produced two survivorsand an MDS of 47.2 days. The 1.0 mg/kg doses had no significantactivity.

[0271] Treatment with 1.5 mg/kg 9-nitro-camptothecin in IDD-P wassuperior to I.P. CAMPTOSAR® (100 mg/kg, qwk×3), HYCAMTIN® (10 mg/kg,q4d×4), and DTIC® (150 mg/kg, qd×5). Each of the latter treatmentsproduced a single CR response and MDS values of 47.3, 46.7, and 37.6days, respectively. 9-nitro-camptothecin in IDD-P appeared lesseffective than oral 9-nitro-camptothecin in 3% DMA (4.0 or 2.0 mg/kg,Days 1, 4, 8, 11), which gave seven survivors, including four CRresponses, and MDS values of 45.3 and 47.6 days, respectively.

[0272] In summary, IDD-P and IDD-D were well tolerated as I.V. vehicles.The anti-melanoma activity of I.V. 9-nitro-camptothecin appeared to begreater in a vehicle containing IDD-P than one with IDD-D. Therapy with1.5 mg/kg 9-nitro-camptothecin in IDD-P was highly effective andappeared superior to optimal I.P. treatments with CAMPTOSAR®, HYCAMTIN®,or DTIC®. 9-nitro-camptothecin in IDD-P appeared less effective at 1.5mg/kg on the 5/2/5 schedule than oral 9-nitro-camptothecin in 3% DMA at2 mg/kg on a q3d×4 schedule, which produced more survivors. The MTD ofI.V.9-nitro-camptothecin in IDD-P is >2 mg/kg and <3 mg/kg on a 5/2/5schedule.

[0273] A. Methods and Materials

[0274] Abbreviations: The following table summarizes the abbreviationsused to describe this study. ABBREVIATIONS CR complete tumor regression,tumor not detected at termination of the study on Day 62 DMAN,N-dimethylacetamide D5W Dextrose 5% in water, pH @ 4.8 I.P.intraperitoneal I.V. intravenous MTD maximum tolerated dose; doseassociated with no more than one death among ten treated mice and lessthan 20% mean body-weight loss p.o. per os, oral PR partial tumorregression, tumor weight on Day 62 lower than on Day 1 qd × 5 1 dose perday for 5 days (Days 1-5) q3d × 4 4 doses delivered as one dose per dayat 3-day intervals q4d × 4 4 doses delivered as one dose per day at4-day intervals qwk × 3 one dose per week for three weeks (Days 1, 8,15) SD/PD survival to 62 days with a stable or progressively increasingtumor weight TGD tumor growth delay, MDS treated-MDS control, theextension of survival time

[0275] Husbandry: Female nude mice were 16 weeks of age (NCR-nufBr;Taconic Farms) for the A375 study. The mice were fed ad libitum water(reverse osmosis, 1 ppm Cl) and autoclaved Prolab NIH-31 M/5% RodentDiet consisting of 18% protein, 5% fat, 6% fiber, and 8% ash. Mice werehoused in static microisolators on a 12-hour light cycle at 21-22° C.(70-72° F.) and 40% -60% humidity. The animal care and use program isAAALAC International accredited and specifically follows and complieswith the recommendation of the National Research Council's Guide for theCare and Use of Laboratory Animals.

[0276] Tumor Implantation: An A375 melanoma fragment (1 mm 3) wasimplanted subcutaneously in the flank of each nude mouse. When thetumors reached the 60-112 mg size range, the mice were sorted intotreatment groups (on Day 1) such that the group mean tumor sizes rangedfrom 71.3 mg -72.5 mg. Estimated tumor weight was calculated using theformula: ${{Tumor}\quad {{Weight}({mg})}} = \frac{w^{2} \times l}{2}$

[0277] where w=width and 1=length in mm of an A375 melanoma.

[0278] Drug Formulations: IDD-P with a mean particle size of 0.07-0.06mm and IDD-D with a mean particle size of 0.15 mm were prepared for thestudy by RTP Pharma Inc. For this, the 9-nitro-camptothecin suspensionswere diluted with water and vehicle to provide doses of 3 mg/kg and 1.5mg/kg in 33% IDD-P, and doses of 2 mg/kg and 1 mg/kg in 100% and 50%IDD-D, respectively. After dilution, 9-nitro-camptothecin suspensionswere mixed thoroughly by gentle agitation and stored at 2-8° C. For itsoral formulation, 9-nitro-camptothecin was dissolved in DMA(N,N-dimethylacetamide, SIGMA) and diluted with D5W to yield a final DMAconcentration of 3%. CAMPTOSAR®, DTIC®, and HYCAMTIN® were obtained asthe marketed drugs and solutions were prepared fresh on the day ofdosing. CAMPTOSAR® (irinotecan, Pharmacia & Upjohn Company, 20 mg/ml)and DTIC® (dacarbazine, Bayer Corporation, 20 mg/ml) were diluted withD5W. HYCAMTIN® (topotecan, SmithKlein Beecham) was reconstituted withwater at 1 mg/ml.

[0279] Treatments: Volumes of 0.20 ml per 20-g mouse, body-weightadjusted, were administered once daily. Tumor-bearing athymic nude micewere matched on Day 1 into 13 groups of ten animals and drugadministration was initiated. The I.V. formulations of9-nitro-camptothecin were given once daily on a 5/2/5 schedule as shownin the protocol summary shown in FIG. 4. Oral 9-nitro-camptothecin doseswere given on a q3d×4 schedule (Days 1, 4, 8, and 11). Three referencedrugs were administered I.P.: CAMPTOSAR® was given in three doses atweekly intervals (qwk×3); HYCAMTIN® was given in four doses at four-dayintervals (q4d×4); and DTIC® was given in five consecutive daily doses(qd×5). The tumor growth control group for these reference treatmentsreceived no treatment (Group 1). The 9-nitro-camptothecin vehicle: 33.3%IDD-P, 100% IDD-D, and 3% DMA, was administered to vehicle controlGroups 2-4, respectively.

[0280] Tumor Growth Endpoint: The tumor growth delay (TGD) method wasused to assess efficacy. Tumor dimensions were measured twice weeklyuntil the experiment was terminated on Day 62. Each animal waseuthanized as a “cancer death” when its A375 melanoma reached a size of2.0 g. Mean Day of Survival (MDS) values for all groups were based onthe calculated day of death of each mouse, which is given by theformula:${{Time}\quad {to}\quad {endpoint}\quad ({calculated})} = {{{Time}\quad {to}\quad {exceed}\quad {endpoint}\quad ({observed})} - \frac{{Wt}_{2} - {{endpoint}\quad {weight}}}{\frac{{Wt}_{2} - {Wt}_{1}}{D_{2} - D_{1}}}}$

[0281] where:

[0282] Time to exceed endpoint (observed)=number of days it takes foreach tumor to grow past the endpoint (cut-off) size. This is the day theanimal is euthanized as a cancer death.

[0283] D₂=day animal is euthanized.

[0284] D₁=last day of caliper measurement before tumor reaches theendpoint.

[0285] Wt₂=tumor weight (mg) on D₂

[0286] Wt₁=tumor weight (mg) on D₁

[0287] Endpoint weight=predetermined “cut-off” tumor size for the modelbeing used.

[0288] TGD values, which represent treatment-effected extensions ofsurvival time, were calculated from MDS_(treated)-MDS_(control), thedifference in MDS value for a given treatment group and the MDS valuefor A375 melanoma-bearing mice that received no treatment or thecorresponding vehicle.

[0289] At the termination of the experiment on Day 62, treatmentresponses in survivors were classified as either complete tumorregression (CR), partial tumor regression (PR), or stable/progressivedisease (SD/PD). A CR response indicates the absence of measurabletumor. In a PR response, the final tumor weight is lower than on Day 1but larger than 0 mg. In a SD/PD response, mice survived to Day 62 witha progressively growing or stable tumor size that had not reached the2.0-g endpoint.

[0290] Evaluation of Toxicity: The maximum tolerated dose (MTD), asdefined by the NCI, is the highest dose at which no more than 10% of theanimals die and the group mean body-weight loss is no more than 20%.Body weights were measured daily for four or five days and then twiceweekly thereafter until the experiments were terminated on Day 62 of thestudy. The animals were also examined frequently for clinical signs ofany adverse, drug-related side effects. Because mice are not tagged forMTD studies, only the mean group body weights recorded in the appendedraw data are significant in those studies.

[0291] Statistics: The unpaired t-test and Mann-Whitney U test(analyzing means and medians, respectively) were employed to determinethe statistical significance of any difference in MDS between atreatment group and the control group, and between different treatmentgroups. Kaplan-Meier plots were constructed and the log-rank test wasused to evaluate the differences in the effects of treatments on TGDoutcome. All analyses for statistical significance were conducted at Plevel of 0.05 (two-tailed). Prism (GraphPad) version 3 was used for thestatistical analyses and graphic presentations.

[0292] B. Results

[0293] The protocols for 13 groups of A375 tumor-bearing mice (n=10) areshown in FIG. 4. The therapeutic and toxic responses for all groups aresummarized in FIG. 5. The individual survival times for the mice in allgroups are presented as a scatter plot in FIG. 6.

[0294] Tumor Growth in the Control Mice: The A375 melanomas in untreatedmice (Group 1) reached the 2.0-g endpoint in nine animals to give a meanday of survival (MDS) of 30.3±4.4 days. The one 62-day survivor withapparently stable/progressive disease (SD/PD) may be taken to representthe background level for somewhat poor tumor takes. The MDS values forvehicle control mice (Groups 2-4) treated I.V. with 33.3% IDD-P, 100%IDD-D, and 3% DMA, respectively, were similar to the MDS of theuntreated control (FIG. 5). The apparent complete remission (CR)response of one mouse in Group 4 may represent a poor tumor take. FIG. 6shows that the survival times of the untreated mice in Group 1 are morewidely scattered than those of the vehicle control mice, Groups 2-4.

[0295] Therapeutic Effects of Intraperitoneal CAMPTOSAR®, HYCAMTIN®, andDTIC®:

[0296] To provide reference standards for the 9-nitro-camptothecintreatments, three groups of mice were treated I.P. with a camptothecinanalog, CAMPTOSAR® or HYCAMTIN®, or with an alkylator, DTIC®, using theoptimal regimen for each drug. The untreated mice in Group 1 were usedas the controls for these reference therapies. Treatment of Group 5 withCAMPTOSAR® at 100 mg/kg qwk×3 produced an MDS of 47.3 days (n=9) and onesurvivor with a CR response. The tumor growth delay (TGD) of 17 days wasstatistically significant (P value of 0.0038, unpaired two-tailedt-test). Treatment of Group 6 with HYCAMTIN® at 10 mg/kg q4d×4 gave anMDS of 46.7 days (n=9), for a statistically significant TGD of 16.4 days(P value of 0.006, t-test). There was one survivor with a CR response.Treatment of Group 7 with DTICS at 150 mg/kg qd×5 produced an MDS of37.6 days (n=8), one CR response, and one toxic death. The TGD of 7.3days was not statistically significant (P value of 0.248, t-test). Giventhe one SD/PD response in the untreated control and the one CR responsein the 3% DMA vehicle control group, it is uncertain whether thesesingle CR responses in Groups 5-7 can be attributed to treatmentefficacy.

[0297] Efficacy of I.V. 9-nitro-camptothecin in IDD-P: The I.V.9-nitro-camptothecin treatments were all administered on a 5/2/5schedule (Days 1-5 and 8-12). Group 2 mice, with an MDS of 29.2±2.5 days(FIG. 3), served as the vehicle control. Ten doses of 3.0 mg/kg9-nitro-camptothecin in IDD-P proved both highly effective and highlytoxic in Group 8 mice. The MDS of 52 days (n=2) gave a significant TGDof 22.8 days (P value of 0.0027, t-test). There was a 50% survival rateand a 30% toxic death rate. There were 1 CR, 3 partial remission (PR),and 1 SD/PD responses recorded on Day 62. The calculated mean tumorweight for Group 8 remained below the Day 1 level until Day 34. This wasthe longest transient PR response recorded for any group in this study.

[0298] At the low dose of 1.5 mg/kg, 9-nitro-camptothecin in IDD-P waswell tolerated by Group 9 mice. The MDS of 50.7 days (n=7) revealed asignificant TGD of 21.5 days (P<0.0001, t-test). The 62-day survivalrate was, however, reduced to 30% in Group 9. There were 2 CR and 1SD/PD responses. The Kaplan-Meier survival curves for untreated mice andmice treated with 9-nitro-camptothecin in IDD-P or vehicle are shown inFIG. 7. When the MDS values for Groups 8 and 9 were compared by t-testanalysis to the MDS for the untreated control, lower P values of 0.0555and 0.0037 were obtained. This reflects the greater dispersion ofsurvival times for Group 1 mice compared to survival times for Group 2.

[0299] Efficacy of I.V 9-nitro-camptothecin in IDD-D: The I.V.treatments with 9-nitro-camptothecin in IDD-D at 2.0 mg/kg and 1.0 mg/kgproduced MDS values of 47.2 days (n=8) and 32.6 days (n=10) in Groups 10and 11, respectively. Group 3 with an MDS of 31.6±3.2 days served as thecontrol. The 15.6-day TGD for the higher dose was statisticallysignificant (P value of 0.005, t-test). Two 62-day survivors, 1 CR and 1PR, were obtained with the 2.0 mg/kg dosing regimen; there were nosurvivors with the 1.0 mg/kg regimen. Kaplan-Meier survival curves foruntreated mice and mice treated with 9-nitro-camptothecin in IDD-D orvehicle are presented in FIG. 8.

[0300] Efficacy of Oral 9-nitro-camptothecin in 3% DMA: The mice inGroups 12 and 13 were treated p.o. with 4.0 and 2.0 mg/kg9-nitro-camptothecin in 3% DMA on a q3d×4 schedule (Days 1, 4, 8, and11). Both therapies were well tolerated and produced 70% survival rates.There were 4 CR and 3 SD/PD responses at the higher dose and 4 CR, 1 RP,and 2 SD/PD responses at the lower dose. Group 4 with an MDS of 26.0±2.3days served as the vehicle control. The MDS values of 45.3 days forGroup 12 (n=3) and 47.6 days for Group 13 (n=3) provided significantTGDs of 19.3 days (P value of 0.0061, t-test) and 21.6 days (P value of0.0007), respectively. FIG. 8 shows the Kaplan-Meier survival curves foruntreated mice and mice treated with oral 9-nitro-catnptothecin in 3%DMA or vehicle.

[0301] Toxicity in this Study: No toxicity was observed in any of thevehicle treatment groups. Three toxic deaths were recorded in Group 8during or after treatment with 3.0 mg/kg 9-nitro-camptothecin in IDD-Pon the 5/2/5 schedule. Two mice were found dead (Days 9 and 15) withbright pink lungs and impacted stomachs. There was evidence of diarrheawith one of the animals. A third mouse was euthanized for poor health onDay 13. This animal had bright pink lungs but an empty stomach andintestines. An acceptable group mean body weight loss of 13.1% was seenin Group 8 on Day 13. One toxic death was recorded on Day 13 in Group 7,which received DTIC®. This animal had lost 30% of its body weight by Day5 and is responsible for most of the 5.2% mean group body-weight lossrecorded for Group 7 on that day. Since none of the other mice showedsignificant body weight changes, this death may represent an event thatis not drug related.

[0302] C. Discussion of Results

[0303] Intravenous administration of isotonic vehicles containing 33.3%IDD-P or 100% IDD-D was found to be well tolerated by athymic nude mice.The formulation of 9-nitro-camptothecin in these vehicles provided ahighly efficacious I.V. treatment for mice bearing the human A375melanoma.

[0304] The particulate 9-nitro-camptothecin suspension in IDD-P appearedto have greater antitumor activity than the droplet suspension in IDD-D.The MDS values were similar for these groups; however, 30% of the Group9 mice survived at the 1.5 mg/kg dose of 9-nitro-camptothecin in IDD-Pwhile only 20% of the Group 10 mice survived at the higher 2.0 mg/kgdose formulated in IDD-D. Comparison of the survival curves for Groups 9and 10 in FIG. 9 illustrates the difference in 62-day survival and thesomewhat higher mean survival of Group 9, as reflected in the relativeposition of the curves. Although the absence of any weight loss in micetreated with the IDD-D formulation indicates low host toxicity, thelimited solubility of 9-nitro-camptothecin in this vehicle precludes anydose escalation. Therefore, 9-nitro-camptothecin in IDD-P appears tohave greater potential as an I.V. antitumor therapy.

[0305]FIG. 10 provides an alternative approach to reviewing andunderstanding the data collected by this study. This figure summarizesthe results of this study in terms of the amount of time (in days) thatelapsed before a tumor in a given mouse reached ten times the initialsize of the tumor. Since the experiment was conducted for 62 days, if atumor did not achieve a ten-fold increase in size before 62 dayselapsed, a 62 day value was employed.

[0306] As can be seen from this figure, a 9-nitro-camptothecinsuspension in IDD-P at 1.5 mg/kg was found to be the most effectivegiven its 100% survival rate. IDD-P at 3 mg/kg was also effective butmore toxic (70% survival). The 9-nitro-camptothecin suspension in IDD-Dwas also found to be effective although less effective than IDD-P.

[0307] Of interesting note, both the IDD-P and IDD-D formulations werefound to be more effective than CAMPTOSAR®, HYCAMTIN®, and DTIC® andwere found to be almost as effective as the oral formulation for9-nitro-camptothecin.

[0308]FIG. 10 provides a comparison of therapeutic results achieved withthe reference I.P. treatments and the optimal 9-nitro-camptothecinregimen in each vehicle. 9-nitro-camptothecin in IDD-P given on the5/2/5 schedule at 1.5 mg/kg produced more survivors than CAMPTOSAR® (0%survivors), HYCAMTIN® (0% survivors), and DTIC® (10% survivors), whenthese reference drugs were given on their optimal regimens. TheKaplan-Meier survival curves for the three reference drugs arepositioned to the left of the curve for 1.5 mg/kg 9-nitro-camptothecinin IDD-P. The 1.5 mg/kg I.V. 9-nitro-camptothecin regimen was lesseffective than 2 mg/kg oral 9-nitro-camptothecin in 3% DMA, even thoughthe I.V. regimen delivered a total dose of 15 mg/kg and the oral dosedelivered a total dose of 8 mg/kg. When oral 9-nitro-camptothecin wasgiven q3d×4 at 2 mg/kg or 4 mg/kg, 70% of the mice survived and 40% wereCR responses. This difference in survival between Groups 9 and 13 is,however, not statistically significant (P value of 0.1138, log-ranktest).

[0309] At the 3 mg/kg dose, I.V. 9-nitro-camptothecin produced 50%survivors and provided the longest period of transient tumor suppressionseen with any treatment group, but was too toxic. It is possible that adose above 1.5 mg/kg but below 3 mg/kg could yield stronger therapeuticactivity without unacceptable toxicity. Alternatively, an intermittentdosing schedule might allow higher doses of 9-nitro-camptothecin inIDD-P to be administered I.V. without increasing host toxicity.Additional studies are required to determine the optimal schedule forthe I.V. administration of 9-nitro-camptothecin in IDD-P formulation.These schedule and dosage issues are being addressed in ongoing studies.

[0310] In summary, 9-nitro-camptothecin in IDD-P was a highly effectivetherapy against human A375 melanoma xenografts in athymic nude mice. Onthe 5/2/5 schedule, 9-nitro-camptothecin in IDD-P appeared to besuperior to the formulation in IDD-D but somewhat less effective thanoral 9-nitro-camptothecin in 3% DMA delivered on a q3d×4 schedule.

[0311] 4. Melanoma Xenograft Model Study 2

[0312] The above described study was repeated using another group ofmice according to the protocol and conditions as described aboveregarding study 1. FIG. 11 summarizes the results of this study in termsof the amount of time (in days) that elapsed before a tumor in a givenmouse reached ten times the initial size of the tumor. Again, since theexperiment was conducted for 62 days, if a tumor did not achieve aten-fold increase in size before 62 days elapsed, a 62 day value wasemployed. As can be seen from the figure, the test results of the firststudy was shown to be largely repeatable. FIGS. 12A-12D provide theindividual mouse data from this study.

[0313] 5. MX-1 Human Breast Cancer Xenograft Study

[0314] The following study evaluates the IDD-P formulation against humanbreast cancer in athymic nude mice. The study used the MX-1 human breastcancer xenograft model to compare the in vivo antitumor efficacy of I.V.9-nitro-camptothecin, in vehicles containing IDD-P and IDD-D to theefficacy of intraperitoneal CAMPTOSAR® and HYCAMTIN®. Except asindicated in the protocol design for this study, as summarized in FIG.13, the study was conducted according to the same procedures asspecified with regard to the above melanoma study.

[0315] In this study, the experiment was conducted for 53 days. If atumor did not achieve a ten-fold increase in size before 53 dayselapsed, a 53 day value was employed.

[0316] As can be seen from FIG. 13, IDD-P was shown to be effective inthe breast cancer model.

[0317] 6. Pan c-Human Pancreatic Cancer Xenoyraft Study

[0318] The following study evaluates the IDD-P formulation againstpancreatic cancer in athymic nude mice. The study used the Pan c-humanpancreatic cancer xenograft model to compare the in vivo antitumorefficacy of I.V. 9-nitro-camptothecin, in vehicles containing IDD-P tothe efficacy of intraperitoneal CAMPTOSAR® and HYCAMTIN®. Except asindicated in the protocol design for this study, as summarized in FIG.14, the study was conducted according to the same procedures asspecified with regard to the above melanoma study.

[0319] In this study, the experiment was conducted for 58 days. If atumor did not achieve a ten-fold increase in size before 58 dayselapsed, a 58 day value was employed.

[0320] As can be seen from FIG. 14, IDD-P was shown to be effective inthe pancreatic cancer model.

[0321] 7. HT-29 Human Colon Cancer XenoLraft Study

[0322] The following study evaluates the IDD-P formulation against coloncancer in athymic nude mice. The study used the HT-29 human colon cancerxenograft model to compare the in vivo antitumor efficacy of I.V.9-nitro-camptothecin, in vehicles containing IDD-P to the efficacy ofintraperitoneal CAMPTOSAR® and HYCAMTIN®. Except as indicated in theprotocol design for this study, as summarized in FIG. 15, the study wasconducted according to the same procedures as specified with regard tothe above melanoma study.

[0323] In this study, the experiment was conducted for 64 days. If atumor did not achieve a ten-fold increase in size before 64 dayselapsed, a 64 day value was employed.

[0324] As can be seen from FIG. 15, both IDD-P was shown to be effectivein the colon cancer model.

[0325] 8. SKMES Human Lune Cancer Xenograft Study

[0326] The following study evaluates the IDD-P formulation against humanlung cancer in athymic nude mice. The study used the SKMES human lungcancer xenograft model to compare the in vivo antitumor efficacy of I.V.9-nitro-camptothecin, in vehicles containing IDD-P to the efficacy ofintraperitoneal CAMPTOSAR® and HYCAMTIN®. Except as indicated in theprotocol design for this study, as summarized in FIG. 16, the study wasconducted according to the same procedures as specified with regard tothe above melanoma study.

[0327] In this study, the experiment was conducted for 61 days. If atumor did not achieve a ten-fold increase in size before 61 dayselapsed, a 61 day value was employed.

[0328] As can be seen from FIG. 16, both IDD-P was shown to be effectivein the lung cancer model.

[0329] While the present invention is disclosed by reference to thepreferred embodiments and examples detailed above, it is to beunderstood that these examples are intended in an illustrative ratherthan limiting sense, as it is contemplated that modifications andcombinations will readily occur to those skilled in the art, whichmodifications and combinations will be within the spirit of theinvention and the scope of the appended claims.

What is claimed is:
 1. A method for administering a camptothecin to apatient comprising: injecting into a patient a pharmaceuticalcomposition comprising an aqueous suspension of solid particles suitablefor intravenous delivery, the solid particles comprising a camptothecin,and a 0.3 nm to 3.0 μm thick layer of a membrane-forming amphipathiclipid wherein the solid particles have mean diameters between about 0.05μm and 10 μm.
 2. A method according to claim 1, wherein thepharmaceutical composition is thermally sterilized prior toadministration.
 3. A method according to claim 1 wherein the patient hasuncontrolled cell proliferation, the camptothecin serving to control thecell proliferation.
 4. A method according to claim 1 wherein the patienthas cancer, the camptothecin serving to treat the cancer.
 5. A methodaccording to claim 1 wherein the camptothecin is selected from the groupconsisting of 9-nitro-20(S)-camptothecin, 9-amino-20(S)-camptothecin,9-methyl-camptothecin, 9-chloro-camptothecin, 9-flouro-camptothecin,7-ethyl camptothecin, 10-methyl-camptothecin, 10-chloro-camptothecin,10-bromo-camptothecin, 10-fluoro-camptothecin, 9-methoxy-camptothecin,11-fluoro-camptothecin, 7-ethyl-10-hydroxy camptothecin,10,11-methylenedioxy camptothecin, and 10,11-ethylenedioxy camptothecin,and 7-(4-methylpiperazinomethylene)-10,11-methylenedioxy camptothecin.6. A method according to claim 1 wherein the camptothecin is selectedfrom the group consisting of 9-nitro-20(S)-camptothecin,9-amino-20(S)-camptothecin,7-ethyl-10-(4-(1-piperdino)-1-piperdino)-carbonyloxy-camptothecin,7-ethyl-10-hydroxy-20(S)-camptothecin,10,11-methylenedioxy-20(S)-camptothecin, 9-chloro-20(S)-camptothecin,9-bromo-20(S)-camptothecin, 9-hydroxy-20(S)-camptothecin, and11-hydroxy-20(S)-camptothecin.
 7. A method according to claim 1 whereinthe camptothecin is 9-nitro-20(S)-camptothecin.
 8. A method according toclaim 1 wherein the pharmaceutical composition has a pH less than
 7. 9.A method according to claim 1 wherein the pharmaceutical composition hasa pH less than
 6. 10. A method according to claim 1 wherein thepharmaceutical composition has a pH between 5 and
 6. 11. A methodaccording to claim 1 wherein the pharmaceutical composition comprises anisotonic solution.
 12. A method according to claim 1 wherein thepharmaceutical composition comprises mannitol or trehalose.
 13. A methodaccording to claim 1 wherein the composition has been thermallysterilized.
 14. A method according to claim 1 wherein the compositionhas been thermally sterilized by heating to at least 121° C. for atleast 15 minutes.
 15. A method according to claim 1 wherein themembrane-forming amphipathic lipid comprises a phospholipid.
 16. Amethod according to claim 15 wherein the phospholipid is selected fromthe group consisting of saturated phospholipids, unsaturatedphospholipids, synthetic phospholipids, natural phospholipids, andcombinations thereof.
 17. A method according to claim 15 wherein thephospholipid is selected from the group consisting of natural andsynthetic lipids, hen egg-derived phospholipid, egg phospholipid,purified egg phospholipid, soy phospholipid, dimyristoyl lecithin,didodecanoyl lecithin, dioeoyl lecithin, dilinoeoyl lecithin,alpha-palmito-beta-oleoyl lecithin, alpha-palmitoyl-beta-linoleoyllecithin, alpha-oleoyl-beta-palmitoyl lecithin, diarachidonyl lecithin,alpha-palmito-beta-myristoyl lecithin, dimyristoyl phosphatidic acid,dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid,phosphatidyl serine, phosphatidyl inositol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidyl glycerol, dioctadecanoyl phosphatidylethanolamine, dioleoyl phosphatidyl ethanolamine, dihexadecylphosphatidyl ethanolamine, dilauryl phosphatidyl ethanolamine,dimyristoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidylethanolamine, Lipoid E80, Lipoid ES, Lipoid 90H, and Lipoid 100H.
 18. Amethod according to claim 15 wherein the phospholipid comprises LipoidE80.
 19. A method according to claim 1 wherein the outer layer furthercomprises cholesterol.
 20. A method according to claim 1 wherein thecamptothecin is present in amounts of up to about 25% w/w.
 21. A methodaccording to claim 1 wherein the camptothecin is present in amounts offrom about 0.05% w/w to about 5% w/w.
 22. A method according to claim 1wherein the camptothecin is present in amounts of from about 0.1% w/w toabout 1% w/w.
 23. A method according to claim 1 wherein the camptothecinis present in amount of about 0.2% w/w.
 24. A method according to claim1 wherein the camptothecin is present in amounts of up to about 5% w/w.25. A method according to claim 1 wherein the membrane-formingamphipathic lipid is present in amounts of from 0.2% w/w to about 5%w/w.
 26. A method according to claim 1 wherein the membrane-formingamphipathic lipid is present in amounts of from 1% w/w to about 5% w/w.27. A method according to claim 1 wherein the membrane-formingamphipathic 2 lipid is present in amounts of about 4% w/w.
 28. A methodfor administering a camptothecin to a patient comprising: injecting intoa patient a pharmaceutical composition comprising a dispersion ofmicrometer to submicrometer size solid particles in an aqueous carriersolution comprising one or more pharmaceutically acceptable tonicitymodifier agents, the solid particles comprising a camptothecin, a firstcoating of not more than 10% w/w of a substantially water-insoluble,pharmaceutically acceptable lipophilic agent in which the camptothecindrug is insoluble or poorly soluble, and a second coating of at leastone membrane-forming surface stabilizing amphipathic lipid, wherein thedispersion does not aggregate, flocculate, or agglomerate, and theparticles do not grow in size above a volume weighted mean diameter of10 μm upon thermal sterilization.
 29. A method for administering acamptothecin to a patient comprising: injecting into a patient apharmaceutical composition comprising a dispersion of micrometer tosubmicrometer size solid particles in an aqueous carrier solutioncomprising one or more pharmaceutically acceptable tonicity modifieragents, the solid particles comprising a camptothecin, and a coating ofat least one membrane-forming surface stabilizing amphipathic lipid,wherein the dispersion does not aggregate, flocculate, or agglomerate,and the particles do not grow in size above a volume weighted meandiameter of 10 μm upon thermal sterilization.
 30. A method foradministering a camptothecin to a patient comprising: injecting into apatient a pharmaceutical composition comprising an aqueous suspension ofparticles having mean diameters between about 0.05 μm and 10 μm, theparticles comprising a camptothecin having an intact lactone ring, amembrane-forming amphiphatic lipid coating an outer surface of theparticle, and one or more tonicity modifying agents, wherein the ratioof lipid to camptothecin in the particle is less than about 150:1 molesper mole, and wherein the mean diameters of the particles increase lessthan 100% upon thermal sterilization.
 31. A method according to claim 30wherein the mean diameters of the particles increase less than 100% uponstorage at 23° C. for at least 1 month.
 32. A method according to claim30 wherein the mean diameters of the particles increase less than 100%upon storage at 23° C. for at least 3 months.
 33. A method according toclaim 28 wherein the lipophilic liquid vehicle is present in amounts upto about 40% w/w.
 34. A method according to claim 28 wherein thelipophilic liquid vehicle is present in amounts up to about 25% w/w. 35.A method according to claim 28 wherein the lipophilic liquid vehicle ispresent in amounts up to about 20% w/w.
 36. A method according to claim28 wherein the lipophilic liquid vehicle is present in amounts greaterthan 0.5% w/w.